Using expression data to fine map QTL associated with fertility in dairy cattle.

BACKGROUND: Female fertility is an important trait in dairy cattle. Identifying putative causal variants associated with fertility may help to improve the accuracy of genomic prediction of fertility. Combining expression data (eQTL) of genes, exons, gene splicing and allele specific expression is a promising approach to fine map QTL to get closer to the causal mutations. Another approach is to identify genomic differences between cows selected for high and low fertility and a selection experiment in New Zealand has created exactly this resource. Our objective was to combine multiple types of expression data, fertility traits and allele frequency in high- (POS) and low-fertility (NEG) cows with a genome-wide association study (GWAS) on calving interval in Australian cows to fine-map QTL associated with fertility in both Australia and New Zealand dairy cattle populations. RESULTS: Variants that were significantly associated with calving interval (CI) were strongly enriched for variants associated with gene, exon, gene splicing and allele-specific expression, indicating that there is substantial overlap between QTL associated with CI and eQTL. We identified 671 genes with significant differential expression between POS and NEG cows, with the largest fold change detected for the CCDC196 gene on chromosome 10. Our results provide numerous candidate genes associated with female fertility in dairy cattle, including GYS2 and TIGAR on chromosome 5 and SYT3 and HSD17B14 on chromosome 18. Multiple QTL regions were located in regions with large numbers of copy number variants (CNV). To identify the causal mutations for these variants, long read sequencing may be useful. CONCLUSIONS: Variants that were significantly associated with CI were highly enriched for eQTL. We detected 671 genes that were differentially expressed between POS and NEG cows. Several QTL detected for CI overlapped with eQTL, providing candidate genes for fertility in dairy cattle.

Comparison of methods for deriving phenotypes from incomplete observation data with an application to age at puberty in dairy cattle.

BACKGROUND: Many phenotypes in animal breeding are derived from incomplete measures, especially if they are challenging or expensive to measure precisely. Examples include time-dependent traits such as reproductive status, or lifespan. Incomplete measures for these traits result in phenotypes that are subject to left-, interval- and right-censoring, where phenotypes are only known to fall below an upper bound, between a lower and upper bound, or above a lower bound respectively. Here we compare three methods for deriving phenotypes from incomplete data using age at first elevation (> 1 ng/mL) in blood plasma progesterone (AGEP4), which generally coincides with onset of puberty, as an example trait. METHODS: We produced AGEP4 phenotypes from three blood samples collected at about 30-day intervals from approximately 5,000 Holstein-Friesian or Holstein-Friesian × Jersey cross-bred dairy heifers managed in 54 seasonal-calving, pasture-based herds in New Zealand. We used these actual data to simulate 7 different visit scenarios, increasing the extent of censoring by disregarding data from one or two of the three visits. Three methods for deriving phenotypes from these data were explored: 1) ordinal categorical variables which were analysed using categorical threshold analysis; 2) continuous variables, with a penalty of 31 d assigned to right-censored phenotypes; and 3) continuous variables, sampled from within a lower and upper bound using a data augmentation approach. RESULTS: Credibility intervals for heritability estimations overlapped across all methods and visit scenarios, but estimated heritabilities tended to be higher when left censoring was reduced. For sires with at least 5 daughters, the correlations between estimated breeding values (EBVs) from our three-visit scenario and each reduced data scenario varied by method, ranging from 0.65 to 0.95. The estimated breed effects also varied by method, but breed differences were smaller as phenotype censoring increased. CONCLUSION: Our results indicate that using some methods, phenotypes derived from one observation per offspring for a time-dependent trait such as AGEP4 may provide comparable sire rankings to three observations per offspring. This has implications for the design of large-scale phenotyping initiatives where animal breeders aim to estimate variance parameters and estimated breeding values (EBVs) for phenotypes that are challenging to measure or prohibitively expensive.

Estimating Heritabilities and Breeding Values From Censored Phenotypes Using a Data Augmentation Approach.

Time-dependent traits are often subject to censorship, where instead of precise phenotypes, only a lower and/or upper bound can be established for some of the individuals. Censorship reduces the precision of phenotypes but can represent compromise between measurement cost and animal ethics considerations. This compromise is particularly relevant for genetic evaluation because phenotyping initiatives often involve thousands of individuals. This research aimed to: 1) demonstrate a data augmentation approach for analysing censored phenotypes, and 2) quantify the implications of phenotype censorship on estimation of heritabilities and predictions of breeding values. First, we simulated uncensored phenotypes, representing fine-scale "age at puberty" for each individual in a population of some 5,000 animals across 50 herds. Analysis of these uncensored phenotypes provided a gold-standard control. We then produced seven "test" phenotypes by superimposing varying degrees of left, interval, and/or right censorship, as if herds were measured on only one, two or three occasions, with a binary measure categorized for animals at each visit (either pre or post pubertal). We demonstrated that our estimates of heritabilities and predictions of breeding values obtained using a data augmentation approach were remarkably robust to phenotype censorship. Our results have important practical implications for measuring time-dependent traits for genetic evaluation. More specifically, we suggest that data collection can be designed with relatively infrequent repeated measures, thereby reducing costs and increasing feasibility across large numbers of animals.

Onset of normal cycles in postpartum anovulatory dairy cattle treated with kisspeptin.

The efficacy of a long-acting synthetic derivative of kisspeptin (Kp) to initiate normal oestrous cycles was tested in 24 mixed-aged, Holstein-Friesian cows that were 18-25 days postpartum on the day of treatment (D0). Groups of eight cows received saline (Sal) vehicle by intramuscular injection at 8:00 and 16:00 h (Sal-Sal), Kp at 8:00 h and vehicle at 16:00 h (Kp-Sal) or Kp on both occasions (Kp-Kp). The Kp dose was 15 nmol per 60 kg body weight. The ovaries of the cows were examined daily by ultrasonography between D4 and D14. Blood samples were collected from a tail vessel at 0, 2, 4, 8, 10 and 12 h relative to the time of the first injection for luteinizing hormone (LH) and follicle-stimulating hormone assay. Additional samples were collected daily from D4 until D14 and D19, 22, 26 and 29 for progesterone assay. LH surge-like responses were observed in cows treated with Kp at 8:00 h. Ovulation was consistently induced by Kp within 48 h when a dominant ovarian follicle of at least 10 mm in diameter was observed (8/14) but in no cases (6/14) during a new wave of ovarian follicular development comprising follicles <10 mm in diameter. The subsequent ovulatory cycle was of normal length in most cases as compared with short 8- to 12-day cycles observed in spontaneously ovulating cows. We conclude that Kp treatment can induce ovulation in postpartum dairy cows, with ensuing oestrous cycles of normal length, if administered when a mature dominant follicle is present in the ovaries. LAY SUMMARY: Cow fertility is important for efficient, profitable dairy farming. Cows that take too long after calving to become fertile are problematic. We tested a synthetically made, long-acting hormone called kisspeptin (Kp) to advance the time that cows become fertile after calving. Twenty-four dairy cows that had been calved for 3-4 weeks were used. One group of eight cows received an injection of Kp at the morning milking, another eight cows received Kp at both the morning and afternoon milking, while the last group of eight cows served as untreated controls. Kp treatment caused a desirable hormone response from the cows' brain. Normal oestrous cycles resulted, but only when a mature follicle was present in the ovary. Further study is required to analyse whether the use of a long-acting Kp drug could be used as an effective treatment for stimulating dairy cows to become more fertile after calving.

Kiss1 expression in the hypothalamic arcuate nucleus is lower in dairy cows of reduced fertility†.

We tested the hypothesis that divergent genetic merit for fertility of dairy cows is due to aberrant reproductive neuroendocrine function. The kisspeptin status of non-pregnant cows of either positive (POS) or negative (NEG) breeding values (BVs) for fertility was studied in three groups (n = 8), based on their previous post-partum period: POS cows, which had spontaneous ovarian cycles (POS-CYC) and NEG cows, which either cycled (NEG-CYC) or did not cycle (NEG-NONCYC). Ovarian cycles were synchronized, blood samples were taken to define endocrine status, and the animals were slaughtered in an artificial follicular phase. The brains and the pituitary glands were collected for quantitative polymerase chain reaction (qPCR) and in situ hybridization of hypothalamic GNRH1, Kiss1, TAC3, and PDYN and pituitary expression of LHB and FSHB. Gonadotropin releasing hormone (GnRH) and kisspeptin levels were quantified in snap frozen median eminence (ME). GNRH1 expression and GnRH levels in the ME were similar across groups. Kiss1 expression in the preoptic area of the hypothalamus was also similar across groups, but Kiss1 in the arcuate nucleus was almost 2-fold higher in POS-CYC cows than in NEG groups. TAC3 expression was higher in POS-CYC cows. The number of pituitary gonadotropes and the level of expression of LHB and FSHB were similar across groups. We conclude that the lower levels of Kiss1 and TAC3 in NEG cows with low fertility status and may lead to deficient GnRH and gonadotropin secretion.

Exosomes from dairy cows of divergent fertility; Action on endometrial cells.

Abnormalities in endometrial function contribute to poor fertility and reproductive failure. Exosomes are small lipid vesicles that contain transferable bioactive substances; they participate in intercellular signaling and may have critical roles in reproductive mechanisms, including endometrial remodeling in preparation for pregnancy. In this study, we evaluated the effects of exosomes from heifers with high and low genetic merit for fertility on inflammatory mediator expression by bovine endometrial epithelial and stromal cell lines. Co-incubation of exosomes from low, compared with high, fertility heifers upregulated the gene expression of pro-inflammatory IL1A and IL8 (CXCL8) but downregulated IL4 gene expression in epithelial cells. In contrast, stromal cells co-incubated with exosomes from low, compared with high, fertility heifers downregulated the gene expression of CXCL9, CXCL10, and CX3CL1. Our findings demonstrated that circulating exosomes from high fertility heifers did not alter endometrial inflammatory mediator gene expression. In contrast, circulating exosomes from low fertility heifers enhanced endometrial expression of inflammatory mediators, which may contribute to aberrant inflammation, leading to a reduced fertility in low fertility heifers. However, an in-depth investigation is required to elucidate the role of exosomes in regulating endometrial remodeling events required for enhanced reproductive performance and fertility in dairy cows.

Circulating exosomes may identify biomarkers for cows at risk for metabolic dysfunction.

Disease susceptibility of dairy cows is greatest during the transition from pregnancy to lactation. Circulating exosomes may provide biomarkers to detect at-risk cows to enhance health and productivity. From 490 cows, animals at high- (n = 20) or low-risk (n = 20) of transition-related diseases were identified using plasma non-esterified fatty acid and ß-hydroxybutyrate concentrations and liver triacylglyceride concentrations during the two weeks post-calving. We isolated circulating exosomes from plasma of dairy cows at low-risk (LR-EXO) and high-risk (HR-EXO), and analyzed their proteome profiles to determine markers for metabolic dysfunction. We evaluated the effects of these exosomes on eicosanoid pathway expression by bovine endometrial stromal (bCSC) and epithelial (bEEL) cells. HR-EXO had significantly lower yield of circulating exosomes compared with LR-EXO, and unique proteins were identified in HR-EXO and LR-EXO. Exposure to LR-EXO or HR-EXO differentially regulated eicosanoid gene expression and production in bCSC and bEEL cells. In bCSC, LR-EXO exposure increased PGE2 and PGD2 production, whereas HR-EXO exposure increased PTGS2 gene expression. In bEEL, HR-EXO exposure caused a decrease in PGE2, PGF2α, PGD2, PGFM and TXB2 production. The unique presence of serpin A3-7, coiled-coil domain containing 88A and inhibin/activin ß A chain in HR-EXO, indicates potential biomarkers for cows at-risk for metabolic diseases. Our results are in line with the health status of the cow indicating a potential diagnostic role for exosomes in enhancing cows' health and fertility.

Proteome profiling of exosomes derived from plasma of heifers with divergent genetic merit for fertility.

The current study evaluated exosomes isolated from plasma of heifers bred to have high or low fertility through developing extreme diversity in fertility breeding values, however, key animal traits (e.g., body weight, milk production, and percentage of North American genetics) remained similar between the 2 groups. The exosomes were isolated by a combined ultracentrifugation and size exclusion chromatography approach and characterized by their size distribution (nanoparticle tracking analysis), morphology (transmission electron microscopy), and presence of exosomal markers (immunoblotting). In addition, a targeted mass spectrometry approach was used to confirm the presence of 2 exosomal markers, tumor susceptibility gene 101 and flotillin 1. The number of exosomes from plasma of high fertility heifers was greater compared with low fertility heifers. Interestingly, the exosomal proteomic profile, evaluated using mass spectrometry, identified 89 and 116 proteins in the high and low fertility heifers respectively, of which 4 and 31 were unique, respectively. These include proteins associated with specific biological processes and molecular functions of fertility. Most notably, the tetratricopeptide repeat protein 41-related, glycodelin, and kelch-like protein 8 were identified in plasma exosomes unique to the low fertility heifers. These proteins are suggested to play a role in reproduction; however, the role of these proteins in dairy cow reproduction remains to be elucidated. Their identification underscores the potential for proteins within exosomes to provide information on the fertility status and physiological condition of the cow. This may potentially lead to the development of prognostic tools and interventions to improving dairy cow fertility.

Proteomic content of circulating exosomes in dairy cows with or without uterine infection.

In the past few decades, there has been a global decrease in dairy cow reproductive performance. An activated inflammatory system, due to uterine infection, has been associated with decreased cow fertility and as such, there is a need to detect uterine disease earlier. Early detection could be achieved by identifying biomarkers for uterine disease. Exosomes are small nanovesicles known to package and deliver protein, mRNA, and miRNAs to near and distant sites. Therefore, the content of circulating exosomes may have the potential to carry biomarkers for earlier diagnosis of disease. We hypothesized that circulating exosomes from cows with and without uterine infection may contain information representative of endometrial health or disease. We compared the proteomic content of circulating exosomes derived from plasma of dairy cows with (n = 10) or without (n = 10) induced uterine infection, using high-performance liquid chromatography tandem mass spectrometry (HPLC MS/MS). Our results demonstrate that there were a total of 103 bovine and 9 Trueperella pyogenes proteins found in plasma exosomes derived from infected cows (infected exosomes), and 90 bovine and 5 T. pyogenes proteins found in exosomes derived from plasma of non-infected cows (non-infected exosomes). 71 bovine proteins were found to be unique to the infected exosomes while only 4 bovine proteins were found to be unique to the non-infected exosomes. 8 unique T. pyogenes proteins were identified in infected exosomes and 4 were found to be unique to the non-infected exosomes. Pathway analysis showed that infected exosomes had more proteins involved in structural molecule activity and immune system processes than non-infected exosomal protein. Additionally, proteins from infected exosomes were involved in unique pathways: angiogenesis and integrin signaling pathway. Our data provide preliminary evidence of a potential role for exosomes in the early diagnosis of uterine infection in dairy cows.

Effect of exosomes from plasma of dairy cows with or without an infected uterus on prostaglandin production by endometrial cell lines.

A contributing factor to declining fertility in dairy cows is an activated inflammatory system associated with uterine infection. Detecting uterine disease using biomarkers may allow earlier diagnosis and intervention with resultant improvements in fertility. Exosomes are known to participate in intercellular communication, paracrine, and endocrine signaling. Exosomes carry a cargo of proteins, lipids, and nucleic acids that represent specific cellular sources. Prostaglandins are lipids that are critical determinants of bovine fertility. In this study exosomes were isolated from the plasma of cows before (d 0) and during (d 10) the study in healthy animals or those with an induced uterine infection in a 2 × 2 factorial design. Exosomes were characterized for size and number (nanoparticle tracking analysis), exosomal marker expression (Western blot), and morphology (transmission electron microscopy). No significant differences were observed in exosome size or number. The abundance of exosome-enriched markers was confirmed in noninfected and infected animals. Transmission electron microscopy confirmed the morphology of the exosomes. These exosomes were co-incubated with bovine endometrial epithelial and stromal cells. Exosomes from d-10-infected animal plasma decreased PGF2α production in endometrial epithelial but not stromal cells. For future research, the identification of effectors in the cargo may provide a useful basis for early diagnosis of uterine infection using an exosomal characterization approach.

Modulation of the immune system during postpartum uterine inflammation.

Postpartum uterine inflammation (endometritis) in the dairy cow is associated with lower fertility at both the time of infection and after the inflammation has resolved. We hypothesized that aberrant DNA methylation may be involved in the subfertility associated with uterine inflammation. The objective of this study was to characterize genome-wide DNA methylation and gene expression in the endometrium of dairy cows with subclinical endometritis (SCE). Endometrial tissues were obtained at 29 days postpartum (n = 12), and microarrays were used to characterize transcription and DNA methylation. Analyses revealed 1,856 probes differentially expressed in animals with SCE (n = 6) compared with controls (CON, n = 6, P < 0.05, Storey Multiple testing correction) and 2,976 probes with significant correlation between gene expression and bacteriology score. No significant associations among DNA methylation and gene expression were detected. Analysis of transcription data using the Database for Annotation, Visualization, and Integrated Discovery and Gene Set Enrichment Analysis identified several pathways and processes enriched in SCE cows, with the majority related to the immune response. Furthermore, the top ontology terms enriched in genes that had expression data correlated to bacteriology score were: Defense response, inflammatory response, and innate immune response. Gene expression profiles in cows with subclinical endometritis in this study indicate that the immune response is activated, potentially resulting in a local proinflammatory environment in the uterus. If this period of inflammation is prolonged it could result in tissue damage or failure to complete involution of the uterus, which may create a suboptimal environment for future pregnancy.

Ovarian activity in Fleckvieh, Brown Swiss and two strains of Holstein-Friesian cows in pasture-based, seasonal calving dairy systems.

The objectives of the study were to compare the ovarian activity of Holstein-Friesian (CH HF), Fleckvieh (CH FV) and Brown Swiss (CH BS) dairy cows of Swiss origin with that of Holstein-Friesian (NZ HF) dairy cows of New Zealand origin, the latter being used as a reference for reproductive performance in pasture-based seasonal calving systems. Fifty, second-lactation NZ HF cows were each paired with a second-lactation Swiss cow (17, 15 and 18 CH HF, CH FV and CH BS respectively) in 13 pasture-based, seasonal-calving commercial dairy farms in Switzerland. Ovarian activity was monitored by progesterone profiling from calving to first breeding service. CH BS cows produced less energy-corrected milk (mean 22·8 kg/d) than the other breeds (26·0-26·5 kg/d) during the first 100 d of lactation. CH HF cows had the lowest body condition score (BCS) at calving and the greatest BCS loss from calving to 30 d post partum. Commencement of luteal activity (CLA) was later for NZ HF than for CH FV (51·5 v. 29·2 d; P <0·01), with CH HF and CH BS intermediate (43 d). On average, NZ HF and CH HF cows had one oestrous cycle before the onset of the seasonal breeding period; this was less (P<0·01) than either CH FV (1·7) or CH BS (1·6). There was a low prevalence of luteal persistency (3%) among the studied cows. First and second oestrous cycle inter-ovulatory intervals did not differ between breeds (20·5-22·6 d). The luteal phase length of CH BS during the second cycle was shorter (10·6 d) than that of the other breeds (13·8-16·0 d), but the inter-luteal interval was longer (9·8 d v. 7·0-8·0 d). The results suggest that the Swiss breeds investigated have a shorter interval from calving to CLA than NZ HF cows.