Project description:The miRNA profiles were measured using small-RNA sequencing in beef heifers sampled at weaning that was retrospectively classified as fertile or subfertile following the breeding protocol. To accomplish this, the miRNA profiles were generated from the blood samples (10 mL) collected from crossbred heifers (Angus-Simmental) at the time of weaning (~238 days after birth). Peripheral white blood cells (PWBC) were extracted from the blood samples and stored at -80°C until further processing. During the breeding season, all the heifers followed the same breeding protocol, estrus synchronization, and fixed-time artificial insemination (AI). Fourteen days following the fixed-time AI, the non-pregnant heifers were exposed to fertile bulls for 60 days. Depending on the presence or absence of conceptus at 75 days following AI, heifers were classified as fertile for those who were pregnant through artificial insemination, pregnant to natural breeding (P-NB), or subfertile for those who were not pregnant. Heifers from fertile (n = 7) and subfertile (n = 7) groups were considered for the study. Total RNA was extracted from the PWBC of 14 samples and was subjected to small RNA library preparation and sequencing. After quality control, adapter trimming, and alignment, mature miRNAs were used for differential expression analysis. The read counts were transformed to counts per million (CPM), and raw counts with CPM < 1 in 50% of the samples were filtered out. The filtered raw counts were analyzed using DESeq2 v 1.26.0 to identify differentially expressed miRNAs (DEMIs). The DEMIs identified with a p-value < 0.05 and absolute (log2 fold change) > 0.5 were considered significant. With the subfertile heifers as the reference group, we identified 16 DEMIs between fertile and subfertile groups. To determine the genes targeting the DEMIs, we downloaded the target genes for each DEMI and retained only those genes expressed in the PWBCs. For the miRNA-gene correlation, we used the partial correlation and information theory (PCIT) approach to identify the significant gene-miRNA correlated pairs. The significant genes correlated with the miRNAs identified pathways including MAPK, ErbB, HIF-1, FoxO, p53, mTOR, T-cell receptor, insulin and GnRH signaling, apoptosis, and pathways regulating pluripotency of stem cells in the fertile group while cell cycle, p53 signaling pathway and apoptosis pathways in the subfertile group.

Project description:Background: Infertility is a longstanding limitation in livestock production with important economic impact for the cattle industry. Female reproductive traits are polygenic and lowly heritable in nature, thus selection for fertility is challenging. Beef cattle operations leverage estrous synchronization in combination with artificial insemination (AI) to breed heifers and benefit from an early and uniform calving season. A couple of weeks following AI, heifers are exposed to bulls for an opportunity to become pregnant by natural breeding (NB), but they may also not become pregnant during this time period. Focusing on beef heifers, in their first breeding season, we hypothesized that: a- at the time of AI, the transcriptome of peripheral white blood cells (PWBC) differs between heifers that become pregnant to AI and heifers that become pregnant late in the breeding season by NB or do not become pregnant during the breeding season; and b- the ratio of transcript abundance between genes in PWBC classifies heifers according to pregnancy by AI, NB, or failure to become pregnant. Results: We generated RNA-sequencing data from 23 heifers from two locations (A: six AI-pregnant and five NB-pregnant; and B: six AI-pregnant and six non-pregnant). After filtering out lowly expressed genes, we quantified transcript abundance for 12,538 genes. The comparison of gene expression levels between AI-pregnant and NB-pregnant heifers yielded 18 differentially expressed genes (DEGs) (ADAM20, ALDH5A1, ANG, BOLA-DQB, DMBT1, FCER1A, GSTM3, KIR3DL1, LOC107131247, LOC618633, LYZ, MNS1, P2RY12, PPP1R1B, SIGLEC14, TPPP, TTLL1, UGT8, eFDR≤0.02). The comparison of gene expression levels between AI-pregnant and non-pregnant heifers yielded six DEGs (ALAS2, CNKSR3, LOC522763, SAXO2, TAC3, TFF2, eFDR≤0.05). We calculated the ratio of expression levels between all gene pairs and assessed their potential to classify samples according to experimental groups. Considering all samples, relative expression from two gene pairs correctly classified 10 out of 12 AI-pregnant heifers (P=0.0028) separately from the other 11 heifers (NB-pregnant, or non-pregnant). Conclusion: The transcriptome profile in PWBC, at the time of AI, is associated with the fertility potential of beef heifers. Transcript levels of specific genes may be further explored as potential classifiers, and thus selection tools, of heifer fertility.

Project description:Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In Study One, 270 predominantly Angus heifers were classified based on fertility using four rounds of serial embryo transfer (ET) to select animals with intrinsic differences in pregnancy loss. In each round, a single in vitro-produced high-quality embryo was transferred into heifers on day 7 post-estrus and pregnancy was determined on days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In Study Two, fertility-classified heifers were resynchronized and bred with semen from a single high fertility bull. Blood samples were collected every other day from days 0 to 36 post-mating. Pregnancy rate was determined on day 28 by ultrasound and tended to be higher in HF (70.4%) and SF (46.7%) than IF (0%) heifers. Progesterone concentrations in serum during the first 20 days post-estrus were not different in non-pregnant heifers and also not different in pregnant heifers among fertility groups. In Study Three, a single in vivo-produced embryo was transferred into fertility-classified heifers on day 7 post-estrus. The uteri were flushed on day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the day 14 endometrial biopsies of pregnant HF, SF and IF heifers (n=5 per group) and analyzed by edgeR robust analysis. There were 26 differentially expressed genes (DEG) in the HF compared to SF endometrium, 12 DEG for SF compared to IF endometrium, and 3 DEG between the HF and IF endometrium. Many of the DEG encoded proteins involved in immune responses and are expressed in B cells. Results indicate that pre-implantation conceptus survival and growth to day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between days 14 and 28 when pregnancy recognition signaling and conceptus implantation must occur for the establishment of pregnancy. Endometrial biopsies were subjected to RNA sequencing from high fertile (HF; n=5), subfertile (SF; n=5) and infertile (IF; n=5) classified heifers on day 14 of pregnancy.

Project description:Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In Study One, 270 predominantly Angus heifers were classified based on fertility using four rounds of serial embryo transfer (ET) to select animals with intrinsic differences in pregnancy loss. In each round, a single in vitro-produced high-quality embryo was transferred into heifers on day 7 post-estrus and pregnancy was determined on days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In Study Two, fertility-classified heifers were resynchronized and bred with semen from a single high fertility bull. Blood samples were collected every other day from days 0 to 36 post-mating. Pregnancy rate was determined on day 28 by ultrasound and tended to be higher in HF (70.4%) and SF (46.7%) than IF (0%) heifers. Progesterone concentrations in serum during the first 20 days post-estrus were not different in non-pregnant heifers and also not different in pregnant heifers among fertility groups. In Study Three, a single in vivo-produced embryo was transferred into fertility-classified heifers on day 7 post-estrus. The uteri were flushed on day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the day 14 endometrial biopsies of pregnant HF, SF and IF heifers (n=5 per group) and analyzed by edgeR robust analysis. There were 26 differentially expressed genes (DEG) in the HF compared to SF endometrium, 12 DEG for SF compared to IF endometrium, and 3 DEG between the HF and IF endometrium. Many of the DEG encoded proteins involved in immune responses and are expressed in B cells. Results indicate that pre-implantation conceptus survival and growth to day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between days 14 and 28 when pregnancy recognition signaling and conceptus implantation must occur for the establishment of pregnancy.

Project description:The gene expression profiles were measured using RNA-Seq beef heifers sampled at weaning and retrospectively classified as pregnant through artificial insemination (AI-P, n = 8) or non-pregnant (NP, n = 7). In brief, crossbred heifers (Angus-Simmental) were used in this study. At the time of weaning (~238 days after birth), blood samples (10 mL) were collected from the jugular vein into vacutainers containing EDTA. The blood was processed to extract PWBC and was stored at -80 until further processing. During the breeding season, all the heifers followed the same breeding protocol, estrus synchronization, and fixed-time artificial insemination (AI). Fourteen days following the fixed-time AI, the non-pregnant heifers were exposed to fertile bulls for 60 days. Depending on the presence or absence of conceptus at 75 days following AI, heifers were classified as pregnant to AI (AI-P), pregnant to natural breeding (P-NB) or non-pregnant (NP). Heifers that were pregnant to AI (N = 8) and non-pregnant (N = 7) were considered for the study. Total RNA was extracted from peripheral white blood cells of 15 samples (AI-P and NP) and was subjected to sequencing. The RNA-Seq data were subjected to quality control and mapped with STAR aligner to Ensemble’s ARS UCD1.2 Bos taurus reference genome. Read quantification was performed using a STAR aligner to obtain raw counts per gene. Genes with low counts were filtered out. After filtering, the read counts were analyzed using DESeq2 v 1.26.0 to identify differentially expressed genes (DEGs). The DEGs with padj < 0.05 and absolute (log2 fold change) > 0.5 were considered significant. The NP heifers were considered as the reference group, and the DEGs up or downregulated were classified based on the log2 fold change direction. We identified 92 DEGs between AI-P and NP groups. The top pathways identified with DEGs were the immune system, cytokine production and defense response, hematopoietic cell lineage, positive regulation of reactive oxygen species metabolic process, and negative regulation of cysteine-type endopeptidase activity involved in the apoptotic process.

Project description:Puberty is a complex physiological event measured by various indicator traits in genetic improvement programs of beef cattle; thus, developing a more complete understanding of the genes and regulatory pathways and networks involved in puberty will provide knowledge to help improve genetic selection strategies. Herein, we characterized the transcriptome of five reproductive tissues (i.e. hypothalamus, pituitary gland, ovary, uterus, and endometrium) as well as tissues known to be relevant to growth and metabolism needed for cattle to achieve puberty (i.e., longissimus dorsi muscle, fat, and liver). These tissues were collected from pre (PRE)- and post (POST)-pubertal Brangus (3/8 Brahman; Bos indicus x 5/8 Angus; Bos taurus) heifers derived from a population of cattle used to identify QTL associated with fertility traits. In order to exploit the power of complementary omics analyses, PRE and POST puberty co-expression gene networks were constructed by combining the results from RNA-Seq, GWAS, and bovine transcription factors. RNA-Seq of 8 tissues among PRE and POST Brangus heifers revealed 1515 differentiallyexpressed and 943 tissue-specific genes within the 17,832 genes confirmed by metrics of RNA-Seq analysis. Combining the results from RNA-Seq and GWAS indentified a total of 25 QTL associated to heifer fertility. The hypothalamus experienced the most notable up-regulation of genes via puberty. Complementary omics procedures revealed 2,450 co-expressed genes across the 8 tissues relative to puberty. The PRE network had 372,861 connections whereas the POST network had 328,357 connections. A sub-network from this process revealed key transcriptional regulators (i.e., PITX2, FOXA1, TSG1D1, DACH2, LHX4, PROP1 and SIX6). Results from multiples sources of omics data will facilitate the design of breeding strategies to improve fertility in Bos indicus-influenced composite cattle. Sixty-one samples from PRE and POST pubertal composite beef heifers were analyzed with RNA-Seq. The transcriptome of five reproductive tissues (i.e. hypothalamus, pituitary gland, ovary, uterus, and endometrium) as well as tissues known to be relevant to metabolism andbody morphometrics needed for cattle to achieve puberty (i.e.,) was characterized. These tissues were collected from pre (PRE)- and post (POST)-pubertal Brangus (3/8 Brahman x 5/8 Angus) heifers derived from a population of cattle used to identify QTL associated with fertility. Total RNA was purified using a Trizol protocol (Invitrogen, Carlsbad, CA). Sequencing libraries were made using TruSeq RNA Sample Preparation kit of Illumina (San Diego, CA).

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Transcriptomics signatures Associated with Pregnancy Status in Beef Heifers

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