Short communication: Validation of in vitro fertility genes in a Holstein bull population

Previously, we constructed an in vitro fertilization system for the identification of genes affecting fertility traits in dairy cattle. The efficiency of this system has been demonstrated by the identification of several genes affecting fertilization rate and early embryonic survival. However, to employ these genetic markers in marker- and gene-assisted selection programs, there is a need to validate in vitro results in phenotypic data sets collected in vivo. Thus, the objective of this study was to validate, in a population of Holstein bulls, the fertility trait genes we previously identified in an in vitro system. Estimated relative conception rate (ERCR) data from 222 Holstein bulls were obtained from 5 different artificial insemination companies in the United States. Bulls were genotyped for the genes FGF2, POU1F1, PRL, PRLR, GH, GHR, STAT5A, OPN, and UTMP, and the data were analyzed for association with ERCR using a mixed effects sire model. A stepwise model selection procedure revealed evidence of association with ERCR for FGF2 and STAT5A polymorphisms. The in vivo validation suggests that these genes can be used in gene-assisted selection programs for reproductive performance in dairy cattle. The genotypes found to be associated with low bull fertility in this study have been reported to be associated with high milk composition in previous studies. These findings provide molecular evidence for the antagonistic relationship between milk production and fertility observed for many years in different breeds of dairy cattle.     

Association of candidate gene polymorphisms with milk technological traits,yield, composition,and somatic cell score in Italian Holstein-Friesian sires

Advances in DNA-based marker technology have enabled the identification of genomic regions underlying complex phenotypic traits in livestock species. The incorporation of detected quantitative trait loci into genetic evaluation provides great potential to enhance selection accuracies, hence expediting the genetic improvement of economically important traits. The objective of the present study was to investigate 96 single nucleotide polymorphisms (SNP) located in 53 candidate genes previously reported to have effects on milk production and quality traits in a population of highly selected Holstein-Friesian bulls. A total of 423 semen samples were used to genotype the bulls through a custom oligo pool assay. Forty-five SNP in 32 genes were found to be associated with at least 1 of the tested traits. Most significant and favorable SNP trait associations were observed for polymorphisms located in CCL3 and AGPAT6 genes for fat yield (0.037 and 0.033 kg/d, respectively), DGKG gene for milk yield (0.698 kg/d), PPARGC1A, CSN1S1, and AGPAT6 genes for fat percentage (0.127, 0.113, and 0.093%, respectively), GHR gene for protein (0.064%) and casein percentage (0.053%), and TLR4 gene for fat (0.090%), protein (0.066%), and casein percentage (0.050%). Somatic cell score was favorably affected by GHR (?0.095) and POU1F1 (?0.137), and interesting SNP-trait associations were observed for polymorphisms located in CSN2, POU1F1, and AGPAT6 genes for rennet coagulation time (?0.592, ?0.558, and ?0.462 min, respectively), and GHR and CSN2 genes for curd firmness 30 min after rennet addition (1.264 and 1.183 mm, respectively). In addition to the influence of individual SNP, the effects of composite genotypes constructed by grouping SNP according to their individual effects on traits considered in the analysis were also examined. Favorable and significant effects on milk traits were observed for 2 composite genotypes, one including 10 SNP and the other 4 SNP. The former was associated with an increase of milk (0.075 kg/d), fat (0.097 kg/d), protein (0.083 kg/d), and casein yields (0.065 kg/d), and the latter was associated with an increase of fat (0.244%), protein (0.071%), and casein percentage (0.047%). Although further research is required to validate the identified SNP loci in other populations and breeds, our results can be considered as a preliminary foundation for further replication studies on gene-assisted selection programs.     

Short communication: Effects of the progesterone receptor variants on fertility traits in cattle

The progesterone receptor (PGR) gene is a key factor in the initiation and maintenance of pregnancy and in embryo development. Currently, it is unknown what variants of the PGR gene are related to fertility traits in cattle. Identification of such variants would allow the implementation of marker-assisted selection in breeding schemes. The objective of this study was to investigate the association of single nucleotide polymorphisms (SNP) of PGR with fertility traits in Holstein dairy cattle. An in vitro fertilization system was used to maximize the efficiency of the identification of genetic factors affecting fertility. This in vitro fertilization system would allow the assessment of fertilization and embryonic survival rates independently of influences from the uterine environment. A total of 5,566 fertilization attempts were performed, and a total of 3,679 embryos were produced using oocytes from 324 Holstein cows and semen from 10 Holstein bulls. Sequencing of pooled DNA samples from ovaries revealed an SNP (G/C) in intron 3 of PGR. A generalized linear model was used to analyze the association of this SNP with fertilization and embryonic survival rates for each ovary. Oocytes obtained from CC ovaries showed a 61% fertilization rate, compared with 68 and 69% for GC and GG ovaries, respectively. The survival rate of embryos produced from GG ovaries was 5 and 6% higher than that of GC and CC ovaries. These results indicate that the PGR SNP could be used in marker-assisted selection breeding programs in Holstein dairy cattle.     

Genome-wide association study and in silico functional analysis of the number of embryos produced by Holstein donors

Superovulation or ovum pick-up and in vitro fertilization are technologies used to produce an increased number of embryos from elite females. Embryo production traits have been shown to be heritable, but the genes that cause this variability have not yet been assessed. The main objectives of this study were to perform a genome-wide association study (GWAS) to find single nucleotide polymorphisms (SNP) associated with embryo production traits and to identify candidate genes affecting the number of embryos produced by Holstein donors in Canada that may provide insight into the regulation of embryo production. Breeding values were estimated and de-regressed for all donors and sires using a data set of 150,971 records of superovulation or ovum pick-up and in vitro fertilization. A total of 11,607 animals were genotyped, but of that number only 5,118 were genotyped with at least a 50K SNP panel and had a de-regressed estimated breeding value reliability of at least 10%. For the GWAS, 606,406 imputed SNP on 29 autosomal chromosomes were considered after applying quality control measures. A single-SNP univariate mixed linear animal model was used to perform the GWAS, and a 5% false discovery rate was applied to adjust for multiple testing. We found 36 and 14 significant SNP associated with the total number of embryos and the number of viable embryos, respectively, with most of them located on chromosome 11. Using these significant SNP, positional genes located within 10,000 bp upstream and downstream of the SNP were retrieved. Thirteen genes were harboring or near the significant SNP for the total number of embryos, 4 of them also being near the significant SNP for viable embryos. Some of these genes (CRB2, DENND1A, MAD1L1, NDUFA8, PTGS1) could be considered as potential positional candidate genes related to the number of embryos produced by a donor. This list will need to be validated in an independent population to confirm the role of the genes for embryo production.     

Association between the GHR,GHRHR, and IGF1 gene polymorphisms and milk yield and quality traits in Sarda sheep

The growth hormone receptor (GHR), the growth hormone releasing hormone receptor (GHRHR), and the insulin-like growth factor 1 (IGF1) genes are known to modulate growth, reproduction, and lactation traits in livestock. The aim of the current work was to investigate if the variation of the sheep GHR, GHRHR, and IGF1 genes is associated with milk yield and quality traits. Three hundred eighty dairy Sarda sheep were genotyped for 36 single nucleotide polymorphisms (SNP) mapping to these 3 loci, and records for milk yield and daily fat and protein yield, as well as for fat, protein, casein, lactose, and milk urea contents, pH, somatic cell score, logarithmic bacterial count, and milk energy were obtained. The linkage disequilibrium analysis was performed only for GHR, as both GHRHR and IGF1 had only 1 polymorphic SNP. Haplotype analysis revealed the existence of 7 haplotype blocks in GHR. Two haplotype blocks, including part of the intron 1 and the upstream region, were clearly separated from the remaining 5 blocks by SNP rs412986330, which may be a recombination hotspot. The latter 5 blocks were contiguous, spanning from intron 2 to exon 10. Statistical analysis revealed that the GHR polymorphism is significantly associated with milk traits for daily fat and protein yield and fat, milk urea, and lactose content. Moreover, variation in IGF1 was associated with milk protein and casein content. Data generated in this research provide new insights into the allelic effects of the ovine GHRHR, GHR, and IGF1 genes on milk production and quality traits, information that may be useful in gene-assisted selection programs.     

Association of lipid-related genes implicated in conceptus elongation with female fertility traits in dairy cattle

Elongation of the preimplantation conceptus is a requirement for pregnancy success in ruminants, and failures in this process are highly associated with subfertility in dairy cattle. Identifying genetic markers that are related to early conceptus development and survival and utilizing these markers in selective breeding can improve the reproductive efficiency of dairy herds. Here, we evaluated the association of 1,679 SNP markers within or close to 183 candidate genes involved in lipid metabolism of the elongating conceptus with different fertility traits in US Holstein cattle. A total of 27,371 bulls with predicted transmitting ability records for daughter pregnancy rate, cow conception rate, and heifer conception rate were used as the discovery population. The associations found in the discovery population were validated using 2 female populations (1,122 heifers and 2,138 lactating cows) each with 4 fertility traits, including success to first insemination, number of services per conception, age at first conception for heifers, or days open for cows. Marker effects were estimated using a linear mixed model with SNP genotype as a linear covariate and a random polygenic effect. After multiple testing correction, 39 SNP flagging 27 candidate genes were associated with at least one fertility trait in the discovery population. Of these 39 markers, 3 SNP were validated in the heifer population and 4 SNP were validated in the cow population. The 3 SNP validated in heifers are located within or near genes CAT, MYOF, and RBP4, and the 4 SNP validated in lactating cows are located within or close to genes CHKA, GNAI1, and HMOX2. These validated genes seem to be relevant for reducing pregnancy losses, and the SNP within these genes are excellent candidates for inclusion in genomic tests to improve reproductive performance in dairy cattle.     

Effect of acute stressors,adrenocorticotropic hormone administration,and cortisol release on milk yield,the expression of key genes,proliferation, and apoptosis in goat mammary epithelial cells

Cortisol is essential to milk synthesis; however, different acute stressors and the exogenous administration of adrenocorticotropic hormone (ACTH) decrease milk yield. Therefore, the effect of cortisol on milk yield and its influence on the survival of mammary epithelial cells have not been fully elucidated. In this context, the objective of this study was to evaluate the effect of cortisol on the expression of growth hormone receptor (GHR), insulin-like growth factor type 1 (IGF1), insulin-like growth factor type 1 receptor (IGF1R), insulin-like growth factor-binding protein 3 and 5 (IGFBP3 and IGFBP5), BAX, and BCL2 genes on the proliferation and apoptotic rates of mammary epithelial cells, and on milk yield in Saanen goats. In the present study, 3 experiments were conducted: (1) comparing the in vivo effects of first milking, vaccination, vermifugation, preventive hoof trimming, and the administration of ACTH or a placebo on cortisol release in dairy goats; (2) studying the in vivo effects of immediate increases in cortisol on the mammary gland of lactating goats; and (3) studying the in vitro effects of a prolonged increase in cortisol on mammary epithelial cells obtained from lactating goats. Cortisol release by goats increased significantly after ACTH administration compared with that observed after a placebo, and the cortisol profiles after first milking, vaccination, vermifugation, hoof trimming, and ACTH administration were similar. However, there was no effect of the immediate increase in cortisol in vivo on IGF-1 release, milk yield, milk quality, or the apoptosis and proliferation rates, nor was there any effect on the expression of the target genes. Furthermore, no interaction was observed between IGF-1 and cortisol in either the in vivo or in vitro experiments. However, the addition of cortisol in vitro significantly increased the expression of the GHR and IGF1R genes, which stimulate cell proliferation, and the BAX gene, which causes apoptosis. These contrasting results can explain why cortisol did not change the rates of proliferation or apoptosis in epithelial cells. Indeed, cortisol supplementation in vitro did not change the number or apoptotic rate of epithelial cells over the course of 5 d. Finally, further studies must be performed to understand the effect of cortisol on the expression of the GHR, IGF1R, and BAX genes by epithelial cells and the roles of these genes in milk synthesis during early lactation.     

Underlying genetic architecture of resistance to mastitis in dairy cattle: A systematic review and gene prioritization analysis of genome-wide association studies

Mastitis, the most frequent disease in dairy cattle. Resistance to mastitis is a complex, polygenic trait controlled by several genes, each with small effects. Genome-wide association studies have been widely used to identify genomic variants associated with complex traits, including resistance to mastitis, to elucidate the underlying genetic architecture of the trait. However, no systematic review and gene prioritization analysis have been conducted to date on GWAS results for resistance to mastitis in dairy cattle. Hence, the objective was to perform a systematic review and gene prioritization analysis of GWAS studies to identify potential functional candidate genes associated with resistance to mastitis-related traits in dairy cattle. Four electronic databases were searched from inception to December 2020, supplemented with multiple sources of gray literature, to identify eligible articles. Annotation for genes and quantitative trait loci (QTL), and QTL enrichment analysis were conducted using GALLO. Gene prioritization analysis was performed by a guilty-by-association approach using GUILDify and ToppGene. From 52 articles included within this systematic review, 30 articles were used for further functional analyses. Gene and QTL annotation resulted in 9,125 and 43,646 unique genes and QTL, respectively, from 39 studies. In general, overlapping of genes across studies was very low (mean ± SD = 0.02% ± 0.07%). Most annotated genes were associated with somatic cell count-related traits and the Holstein breed. Within all annotated genes, 74 genes were shared among Holstein, Jersey, and Ayrshire breeds. Approximately 7.5% of annotated QTL were related to QTL class “health.” Within the health QTL class, 2.6 and 2.2% of QTL were associated with clinical mastitis and somatic cell count-related traits. Enrichment analysis of QTL demonstrated that many enriched QTL were associated with somatic cell score located in Bos taurus autosomes 5, 6, 16, and 20. The prioritization analysis resulted in 427 significant genes after multiple test correction (false discovery rate of 5%) from 26 studies. Most prioritized genes were located in Bos taurus autosomes 19 and 7, and most top-ranked genes were from the cytokine superfamily (e.g., chemokines, interleukins, transforming growth factors, and tumor necrosis factor genes). Although most prioritized genes (397) were associated with somatic cell count-related traits, only 54 genes were associated with clinical mastitis-related traits. Twenty-four genes (ABCC9, ACHE, ADCYAP1, ARC, BCL2L1, CDKN1A, EPO, GABBR2, GDNF, GNRHR, IKBKE, JAG1, KCNJ8, KCNQ1, LIFR, MC3R, MYOZ3, NFKB1, OSMR, PPP3CA, PRLR, SHARPIN, SLC1A3, and TNFRSF25) were reported for both somatic cell count and clinical mastitis-related traits. Prioritized genes were mainly associated with immune response, regulation of secretion, locomotion, cell proliferation, and development. In conclusion, this study provided a fine-mapping of previously identified genomic regions associated with resistance to mastitis and identified key functional candidate genes for resistance to mastitis, which can be used to develop enhanced genomic strategies to combat mastitis by increasing mastitis resistance through genetic selection.     

Genome-wide association study for milk production and conformation traits in Canadian Alpine and Saanen dairy goats

Increasing the productivity of Canadian dairy goats is critical to the competitiveness of the sector; however, little is known about the underlying genetic architecture of economically important traits in these populations. Consequently, the objectives of this study were as follows: (1) to perform a single-step GWAS for milk production traits (milk, protein, and fat yields, and protein and fat percentages in first and later lactations) and conformation traits (body capacity, dairy character, feet and legs, fore udder, general appearance, rear udder, suspensory ligament, and teats) in the Canadian Alpine and Saanen breeds; and (2) to identify positional and functional candidate genes related to these traits. The data available for analysis included 305-d milk production records for 6,409 Alpine and 3,434 Saanen does in first lactation and 5,827 Alpine and 2,632 Saanen does in later lactations; as well as linear type conformation records for 5,158 Alpine and 2,342 Saanen does. Genotypes were available for 833 Alpine and 874 Saanen animals. Both single-breed and multiple-breed GWAS were performed using single-trait animal models. Positional and functional candidate genes were then identified in downstream analyses. The GWAS identified 189 unique SNP that were significant at the chromosomal level, corresponding to 271 unique positional candidate genes within 50 kb up- and downstream, across breeds and traits. This study provides evidence for the economic importance of several candidate genes (e.g., CSN1S1, CSN2, CSN1S2, CSN3, DGAT1, and ZNF16) in the Canadian Alpine and Saanen populations that have been previously reported in other dairy goat populations. Moreover, several novel positional and functional candidate genes (e.g., RPL8, DCK, and MOB1B) were also identified. Overall, the results of this study have provided greater insight into the genetic architecture of milk production and conformation traits in the Canadian Alpine and Saanen populations. Greater understanding of these traits will help to improve dairy goat breeding programs.     

Acute and chronic effects of cortisol on milk yield,the expression of key receptors,and apoptosis of mammary epithelial cells in Saanen goats

Cortisol (CORT) induces mammary development in late gestation and is fundamental to the differentiation of mammary epithelial cells and lactogenesis. The objective of this study was to investigate the relationship between CORT, insulin, prolactin, growth hormone, and insulin-like growth factor-1 in milk as well as the effect of CORT on the expression of receptors of insulin (INSR), prolactin (PRLR), growth hormone (GHR); we also studied the insulin-like growth factor-1 (IGF1R), glucocorticoid (NR3C1), mineralocorticoid (NR3C2), B-cell lymphoma 2 (BCL2), BCL-2-like protein X (BAX) genes, and the apoptosis rate of mammary epithelial cells of lactating Saanen goats in vivo and in vitro. The following experiments were conducted: (1) comparing hormone release in milk and blood after ACTH or a placebo administration; (2) evaluating the effect of acute CORT increases in mammary gland expression and milk yield in vivo; and (3) evaluating the effect of a chronic increase in CORT concentration in epithelial mammary cell apoptosis in vitro. In vivo, ACTH administration significantly increased CORT release but did not affect insulin, prolactin, growth hormone, and insulin-like growth factor-1 release in plasma and milk versus placebo. The results show also that a low CORT release after ACTH administration increased the expression of GHR and PRLR genes in the mammary tissue. Indeed, CORT release significantly increased the milk yield from goats subjected to ACTH versus goats subjected to the placebo. However, a higher amount of CORT added in vitro upregulated the NR3C1, GHR, PRLR, and BAX genes and downregulated the IGF1R and INSR genes, which could negatively modulate the apoptosis of mammary epithelial cells. Finally, the effect of CORT in vivo after ACTH administration demonstrated the increased expression of the PRLR and GHR genes, which may improve epithelial cell responsiveness and be associated with the positive effect of CORT observed on milk yield at mid-end lactation.     

Exploring genome-wide differentiation and signatures of selection in Italian and North American Holstein populations

Among Italian dairy cattle, the Holstein is the most reared breed for the production of Parmigiano Reggiano protected designation of origin cheese, which represents one of the most renowned products in the entire Italian dairy industry. In this work, we used a medium-density genome-wide data set consisting of 79,464 imputed SNPs to study the genetic structure of Italian Holstein breed, including the population reared in the area of Parmigiano Reggiano cheese production, and assessing its distinctiveness from the North American population. Multidimensional scaling and ADMIXTURE approaches were used to explore the genetic structure among populations. We also investigated putative genomic regions under selection among these 3 populations by combining 4 different statistical methods based either on allele frequencies (single marker and window-based) or extended haplotype homozygosity (EHH; standardized log-ratio of integrated EHH and cross-population EHH). The genetic structure results allowed us to clearly distinguish the 3 Holstein populations; however, the most remarkable difference was observed between Italian and North American stock. Selection signature analyses identified several significant SNPs falling within or closer to genes with known roles in several traits such as milk quality, resistance to disease, and fertility. In particular, a total of 22 genes related to milk production have been identified using the 2 allele frequency approaches. Among these, a convergent signal has been found in the VPS8 gene which resulted to be involved in milk traits, whereas other genes (CYP7B1, KSR2, C4A, LIPE, DCDC1, GPR20, and ST3GAL1) resulted to be associated with quantitative trait loci related to milk yield and composition in terms of fat and protein percentage. In contrast, a total of 7 genomic regions were identified combining the results of standardized log-ratio of integrated EHH and cross-population EHH. In these regions candidate genes for milk traits were also identified. Moreover, this was also confirmed by the enrichment analyses in which we found that the majority of the significantly enriched quantitative trait loci were linked to milk traits, whereas the gene ontology and pathway enrichment analysis pointed to molecular functions and biological processes involved in AA transmembrane transport and methane metabolism pathway. This study provides information on the genetic structure of the examined populations, showing that they are distinguishable from each other. Furthermore, the selection signature analyses can be considered as a starting point for future studies in the identification of causal mutations and consequent implementation of more practical application.     

In vitro maturation, fertilization and embryo development after ultrarapid freezing of immature human oocytes

The purpose of this study was to determine the rates of maturation, fertilization and embryo development of ultrarapidly frozen immature oocytes (immature cumulus-oocyte complexes; COCs) obtained from antral follicles in ovaries of patients with chocolate ovarian cysts. The COCs were cryopreserved by a vitrification method using 5.5 mol ethylene glycol l (-1) plus 1.0 mol sucrose l (-1) in Dulbecco's PBS (DPBS). The survival, maturation and fertilization rates, and the percentage of embryos developing to the two-cell stage were 59, 64, 70 and 71%, respectively. No significant differences were noted in the rates of maturation, fertilization and embryo development between control and cryopreserved oocytes. Two embryos that developed from cryopreserved oocytes of the oocyte donor programme were selected for transfer into the uterus of a recipient with premature ovarian failure, after the recipient had received steroid replacement. A biochemical pregnancy occurred in the recipient after embryo transfer. These results indicate that immature oocytes can survive after cryopreservation and subsequently can be cultured to mature oocytes that are capable of undergoing fertilization in vitro and developing into embryos.     

Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts

Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured before in vitro fertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.     

Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos

Accumulating studies have suggested that microRNA play a part in regulating multiple cellular processes, such as cell proliferation, apoptosis, the cell cycle, and embryo development. This study explored the effects of miR-101-2 on donor cell physiological status and the development of Holstein cow somatic cell nuclear transfer (SCNT) embryos in vitro. Holstein cow bovine fetal fibroblasts (BFF) overexpressing miR-101-2 were used as donor cells to perform SCNT; then, cleavage rate, blastocyst rate, inner cell mass-to-trophectoderm ratio, and the expression of some development- and apoptosis-related genes in different groups were analyzed. The miR-101-2 suppressed the expression of inhibitor of growth protein 3 (ING3) at mRNA and protein levels, expedited cell proliferation, and decreased apoptosis in BFF, suggesting that ING3, a target gene of miR-101-2, is a potential player in this process. Moreover, by utilizing donor cells overexpressing miR-101-2, the development of bovine SCNT embryos in vitro was significantly enhanced; the apoptotic rate in SCNT blastocysts was reduced, and the inner cell mass-to-trophectoderm ratio and SOX2, POU5F1, and BCL2L1 expression significantly increased, whereas BAX and ING3 expression decreased. Collectively, these findings suggest that miR-101-2 promotes BFF proliferation and vitality, reduces their apoptosis, and improves the early development of SCNT embryos.     

Longitudinal genome-wide association studies of milk production traits in Holstein cattle using whole-genome sequence data imputed from medium-density chip data

Longitudinal traits, such as milk production traits in dairy cattle, are featured by having phenotypic values at multiple time points, which change dynamically over time. In this study, we first imputed SNP chip (50–100K) data to whole-genome sequence (WGS) data in a Chinese Holstein population consisting of 6,470 cows. The imputation accuracies were 0.88 to 0.97 on average after quality control. We then performed longitudinal GWAS in this population based on a random regression test-day model using the imputed WGS data. The longitudinal GWAS revealed 16, 39, and 75 quantitative trait locus regions associated with milk yield, fat percentage, and protein percentage, respectively. We estimated the 95% confidence intervals (CI) for these quantitative trait locus regions using the logP drop method and identified 581 genes involved in these CI. Further, we focused on the CI that covered or overlapped with only 1 gene or the CI that contained an extremely significant top SNP. Twenty-eight candidate genes were identified in these CI. Most of them have been reported in the literature to be associated with milk production traits, such as DGAT1, HSF1, MGST1, GHR, ABCG2, ADCK5, and CSN1S1. Among the unreported novel genes, some also showed good potential as candidate genes, such as CCSER1, CUX2, SNTB1, RGS7, OSR2, and STK3, and are worth being further investigated. Our study provided not only new insights into the candidate genes for milk production traits, but also a general framework for longitudinal GWAS based on random regression test-day model using WGS data.     

Testing candidate gene effects on milk production traits in dairy cattle under various parameterizations and modes of inheritance

The major objectives of this study were 1) to assess the statistical properties of models commonly used for the estimation of single nucleotide polymorphism (SNP) effects under the assumption of various modes of inheritance and various parameterizations of SNP genotypes using simulated data, and 2) to compare effects of the selected polymorphisms located within butyrophilin (BTN1A1), diacylglycerol acyltransferase 1 (DGAT1), leptin (LEP), and leptin receptor (LEPR) candidate genes on milk production traits using data from 2 dairy cattle breeds (190 Jersey cows and 475 Polish Holstein-Friesian cows). Simulation results showed that type I error and power were not dependent on the assumed parameterization, but differences were observed regarding confidence intervals of estimated SNP effects. In the presence of epistasis, correct confidence intervals for all (epistatic and nonepistatic) SNP and all modes of inheritance were provided only by the parameterization proposed by C. H. Kao and Z. B. Zeng in 2002. However, if no dominance effect was included in the model, confidence intervals for SNP effects were correct for all parameterizations. Results based on real data showed that for both breeds the additive effects of polymorphisms were generally similar, except for LEPR, which had a different allele associated with increased fat content in Holstein-Friesians than in Jerseys. In both breeds, DGAT1 had the largest additive effect of the polymorphisms considered, but its effect on most milk traits was more pronounced in Jerseys than in Holstein-Friesians. Evidence of epistasis was found between LEPR and DGAT1, as well as between LEPR and BTN1A1, but only for milk content traits and only in the Holstein-Friesian breed. There was also more evidence for dominance in the Holstein-Friesian breed than in the Jersey breed.