Progress 04/01/20 to 03/31/23
Outputs
Target Audience:Target audiences for this project include farmers and sheep producers in Wisconsin, undergraduate students, and the general scientific community. Efforts to deliver the outcomes of this research included the following: 1. Presentations at the Annual Sheep Day organized by the Sheep Unit in the Arlington Research Station, Arlington, Wisconsin. 2. Training of several undergraduate students on the effects of nutrition on sheep production and reproduction traits. 3. Integration of research project results into the undergraduate course "Veterinary Genetics" taught by the project director. 4. Peer-reviewed publications and invited presentations to the scientific community as described in the following sections. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project offered an excellent opportunity to train undergraduate students, graduate students, and post-doctoral fellows. -Blake Johnson is an undergraduate student who helped with sheep management, semen collection, and growth and reproductive performance traits collection. -Julian Useche Paredes: Julian is an undergraduate student who was trained in molecular aspects of the project, including gene expression and DNA methylation analyses. -Nicole Gross: a graduate student trained in molecular genetics aspects, including gene expression, DNA methylation, and bioinformatics analysis. Nicole's study was published in Frontiers in Genetics journal in 2020. -Jessica Townsend: a graduate in my lab who worked on the effects of paternal nutrition on the reproductive performance of the offspring. Her study was published in 2022 (Townsend et al., 2022). -Camila Braz: a post-doctoral fellow who collected the data on the effects of paternal nutrition on the production and reproduction traits of F1 and F2 generations. She analyzed the DNA methylation profiles of these generations. Camila's results were published in the PNAS-Nexus journal in 2022 (Braz et al., 2022). How have the results been disseminated to communities of interest?In addition to the scientific publications and invited presentations to the scientific community, the results were disseminated to other communities, such as sheep producers and farmers in Wisconsin and the Netherlands: Wisconsin The effects of paternal nutrition on traits of the offspring in sheep. 12th Annual Arlington Sheep Day. Arlington Agricultural Research Station. Wisconsin. April 2, 2022. The Netherlands Transgenerational epigenetics inheritance in sheep. The 2023 VEERkracht Dairy Congress. The Netherlands. January 30, 2023. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Specific Objective 1: To assess the impact of the paternal diet on reproduction, growth, and carcass traits of offspring in the subsequent three generations. Accomplishments For this specific objective, we aimed to evaluate the impacts of the pre-pubertal diet in Polypay rams (a composite sheep breed constituted in the USA in the late 1960s) on production and reproduction traits in the subsequent generations. The paternal genome's susceptibility to epigenetic modifications increases during specific critical development windows, such as puberty. Therefore, from weaning until puberty, rams were separated once daily for diet administration. Half of the rams (12 twin rams) were fed the control diet (basal concentrate diet of the farm), and the other half (12 twin rams) were fed a treatment diet (basal concentrate diet plus 0.22% added rumen-protected methionine). Methionine-treated rams reached puberty 1.5 weeks earlier than control rams (P = 0.03). Also, treatment animals at puberty were 2.2 kg lighter than controls. Ten rams were chosen for breeding to produce the F1 generation. The diet of F0 rams was associated with scrotal circumference (P = 0.007) and weight at puberty (P = 0.04) of F1 offspring. Interestingly, the methionine-treated littermate rams had 15.3% more males per litter than their control littermates (P = 0.05). These results were published by Gross et al. (2020). The concept of transgenerational epigenetic inheritance (TEI) implies that environmentally-induced epigenetic changes are transmitted to unexposed subsequent generations and associated with transgenerationally-inherited phenotypes. Therefore, we evaluated whether the F0 generation methionine-supplemented diet affected the growth and reproduction phenotypes of the F1 and F2 generations. Testicular size (measured as scrotal circumference) was significantly associated with the F0 diet in both F1 (P = 0.032) and F2 (P = 0.049) generations. The males of the methionine-treated F1 and F2 generations, that were not themselves fed the methionine diet, had 1.00 and 0.81 cm smaller scrotal circumference, respectively, when compared with control groups. Testicular size is positively correlated with male fertility and is considered an indirect measure of testicular functions and spermatogenesis. The growth traits of weaning weight (WWT) and post-weaning weight (PWT) were significantly affected by F0 paternal diet only in F2 generation females (P = 0.020, P = 0.029, respectively). Similarly, loin muscle depth (LMD), a growth-related phenotype, was significant (P = 0.003) in males of the F2 generation. These results were published in PNAS-Nexus in 2022 (Braz et al., 2022). Overall, this study demonstrated that paternal methionine supplementation affected the offspring's phenotypes, although these offspring were not exposed to the paternal diet. Specific Objective 2: To elucidate the epigenetic mechanisms by which paternal diet affects traits of the offspring. Accomplishments To examine whether methionine supplementation alters DNA methylation patterns in sperm of F0 generation, we performed whole-genome bisulfite sequencing (WGBS) for the 10 F0 males used to produce the F1 generation. A total of 5,669 differentially methylated cytosines (DMCs), defined as cytosines with methylation level difference ≥ 20% and false discovery rate (FDR) ≤ 0.01, in the CG context (dinucleotide where a cytosine is followed by a guanine base), were found between the treatment and the control groups. We also found DMCs in non-CG contexts, including 1,288 CHHs (trinucleotides where H represents A, C, or T) and 329 CHGs. To investigate whether the DMCs of the F0 generation are inherited transgenerationally, we performed WGBS of sperm from 45 F1 animals and 20 sperm samples from the F2 generation (10 grand-progeny from the F0 treated group and 10 grand-progeny from the F0 control group). There was a significant overlap of sperm DMCs between the F0 and F1 generations, including 839 CGs, 139 CHHs, and 34 CHGs (Braz et al., 2022). The use of WGBS allowed us to investigate TEI at the single-nucleotide resolution level and identify a remarkable consistency of paternal diet-induced DMCs across three generations compared to previous studies where low-resolution DNA methylation methods were used. Here we show, for the first time, that environmentally-altered epigenetic marks in sheep are transmitted to subsequent generations that were not directly exposed to the diet that initiated the epigenetic changes. Over 100 methylated cytosines, environmentally altered in the F0 generation, were inherited by the F1 and F2 generations. These results improve our understanding of mechanisms of non-Mendelian inheritance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Townsend J, Braz CU, Taylor T, Khatib H. Effects of paternal methionine supplementation on sperm DNA methylation and embryo transcriptome in sheep. Environ Epigenet. 2022 Dec 23;9(1):dvac029. doi: 10.1093/eep/dvac029. PMID: 36727109; PMCID: PMC9885981.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Braz CU, Taylor T, Namous H, Townsend J, Crenshaw T, Khatib H. Paternal diet induces transgenerational epigenetic inheritance of DNA methylation signatures and phenotypes in sheep model. PNAS Nexus. 2022 Apr 14;1(2):pgac040. doi: 10.1093/pnasnexus/pgac040. PMID: 36713326; PMCID: PMC9802161.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Khatib H. 2021. Transgenerational epigenetic inheritance in farm animals: How substantial is the evidence?. Livestock Science 250, 104557, https://doi.org/10.1016/j.livsci.2021.104557
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Environmental epigenomics and transgenerational epigenetic inheritance in livestock and shellfish species. The 115th annual meeting of the National Shellfisheries Association, Baltimore, Maryland. March 30, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Epigenetics and the Gene-Environment Interaction Connection: What We've Learned. Quantitative Genetics and Genomics, the Gordon Research Conference, Ventura, CA. February 13, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Transgenerational epigenetics inheritance in sheep. The 2023 VEERkracht Dairy Congress. The Netherlands. January 30, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Effects of paternal nutrition on the epigenome and phenotypes of the offspring. The 2022 Aspen Perinatal Biology Symposium. Aspen, Colorado. August 28-31, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Environmental Epigenetics and transgenerational epigenetic inheritance. First International Symposium on ONE HEALTH Epigenomes and Microbiomes: From Soil to People. Quito, Ecuador. September 13, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Effects of maternal and paternal nutrition on traits of the next generations. First International Symposium on ONE HEALTH Epigenomes and Microbiomes: From Soil to People. Quito, Ecuador. September 14, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Transgenerational Epigenetic Inheritance in Mammals:
How Strong is the Evidence?. The Epigenetics Symposium, The University of Wisconsin-Madison. October 13, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
The effects of paternal nutrition on traits of the offspring in sheep. 12th Annual Arlington Sheep Day. Arlington Agricultural Research Station. Wisconsin. April 2, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
The effects of parental nutrition on phenotypes of the next generations. The International Conference on Beef Cattle Improvement and Industrialization (6th Edition), Yangling, China. November 26-28, 2021.
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Progress 04/01/21 to 03/31/22
Outputs
Target Audience:The target audience in the previous year was the scientific community. A paper about transgenerational epigenetic inheritance was published in the Livestock Science journal. Khatib H* (2021) Transgenerational epigenetic inheritance in farm animals: How substantial is the evidence? Livestock Science 250, 104557, https://doi.org/10.1016/j.livsci.2021.104557. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided an excellent opportunity to train a minority undergraduate student in my lab. The student helped with farm data collection and lab work such as DNA and RNA extractions from semen samples. Also, the post-doctoral fellow was trained for bioinformatics analysis of the RNA-Seq and whole-genome DNA methylation data. How have the results been disseminated to communities of interest?The results of the studies in the first year of the project were disseminated to the scientific community by publishing a paper in Frontiers in Genetics (Gross et al. 2020). Another paper was published in the Livestock Science journal in 2021. Another manuscript has recently been submitted and it is currently under review. What do you plan to do during the next reporting period to accomplish the goals?During the next year, we will focus on the DNA methylation analysis of the F3 generation. Also, we will collect phenotypic data from the F4 generation.
Impacts
What was accomplished under these goals?
Objective 1: Phenotypic data including growth, body weight, scrotal circumference and semen quality traits have been collected from the F1, F2, and F3 generations from the methionine treatment and the control animals. To evaluate whether the F0 methionine-supplemented diet affected growth and reproduction phenotypes of the F1 and F2 generations, we performed an association analysis between F0 diet and birth weight (BW), weaning weight (WW), post-weaning weight (PWW), scrotal circumference (SC), fat depth (FD), and loin muscle depth (LMD) phenotypes. Testicular size (measured as scrotal circumference) was significantly associated with the F0 diet in both F1 (P = 0.032) and F2 (P = 0.049) generations. The males of the methionine F1 and F2 generations that were not themselves fed the methionine diet had, on average, 1.00 and 0.81 cm smaller scrotal circumference, respectively, when compared with the control groups. Testicular size is positively correlated with male fertility and is considered an indirect measure of testicular functions and spermatogenesis. The growth traits of WWT and PWT were significantly affected by F0 paternal diet only in F2 generation females (P = 0.020, P = 0.029, respectively). Similarly, loin muscle depth, a growth-related phenotype, was significant (P = 0.003) in males of the F2 generation. Objective 2: Whole-genome DNA methylation using bisulfite sequencing at the single nucleotide level has been performed for F1 and F2 generations. Data were analyzed as well. To examine whether methionine supplementation alters DNA methylation patterns in sperm, we performed whole-genome bisulfite sequencing (WGBS) for the 10 F0 males used to produce the F1 generation. A total of 5,669 differentially methylated cytosines (DMCs) in the CG context (methylation level difference ≥ 20% and false discovery rate [FDR] ≤ 0.01) were found between the treatment and the control groups. We also found DMCs in non-CG contexts, including 1,288 CHHs (where H is A, C, or T) and 329 CHGs. To investigate whether the DMCs of the F0 generation are inherited transgenerationally, we performed WGBS of sperm from 45 F1 animals and 20 individual sperm samples of males from the F2 generation. We detected 2,911 CG, 451 CHH, 121 CHG DMCs in the F1 generation and 2,661 CG, 1,553 CHH, and 416 CHG DMCs in the F2 generation. Then, we assessed whether sperm DMCs were present across all three generations. Surprisingly, we identified 107 transgenerationally inherited modified cytosines; 82, 20, and 5 DMCs were in CG, CHH, and CHG contexts, respectively. We further investigated whether the hypermethylation and hypomethylation trends of these DMCs were maintained in the treatment groups from the F0 to the F2 generation. Interestingly, 96 of 107 transgenerationally inherited cytosines (89.7%; 72 CGs, 19 CHHs, and 5 CHGs) demonstrated the same trend of hypermethylation or hypomethylation in the treatment groups across three generations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Khatib, H. (2021). Transgenerational epigenetic inheritance in farm animals: How substantial is the evidence?. Livestock Science 250, 104557, https://doi.org/10.1016/j.livsci.2021.104557.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Nature vs. nurture: what do we inherit from our parents? The Department of Animal Sciences, University of Maryland. April 13, 2021
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