Tissue-Specific Mitochondrial Functionality and Mitochondrial-Related Gene Profiles in Response to Maternal Nutrition and One-Carbon Metabolite Supplementation During Early Pregnancy in Heifers

This case study summarizes our published work in Animals (2025). Read the full paper: https://doi.org/10.3390/ani15182689

Highlights

• 29 crossbred Angus heifers carrying female fetuses were fed for either moderate gain or low gain (slight weight loss) from breeding till day 63 of gestation, factored with or without one-carbon metabolites (methionine, choline, folate, and vitamin B12).
• Fetal liver and muscle were collected at day 161, roughly mid-gestation, long after treatments had stopped.
• In fetal liver, supplementation increased mitochondrial respiration in well-fed heifers and increased mtDNA copy number.
• Mitochondrial respiration in fetal muscle was not influenced, but down-regulated a set of genes associated with fat metabolism and energy regulation.
• About 90% of the mitochondria-related gene changes were in muscle, only 10% in liver.

Methodology

The study used a 2 × 2 factorial experimental design. Control heifers were targeted to gain 0.45 kg a day while restricted heifers to lose 0.23 kg a day. Supplemented heifers got rumen-protected methionine and choline in feed plus weekly injections of vitamin B12 and folic acid. From day 63, every heifer received the same control diet.

At day 161, we collected fetal liver and semitendinosus muscle and evaluated these tissues for oxygen use by isolating mitochondria and analyzing them through a Seahorse XFe24 analyzer, mtDNA copy number by qPCR (ND2 and COX3), and gene expression by RNA-seq. This trial belongs to the NDSU heifer work on maternal diet listed on my research page.

Results

• Fetal liver: state 3 respiration depended on the gain × supplement interaction (p = 0.004). It was highest in control + supplement fetuses.
• Leak (state 4o) respiration in liver was lowest in restricted + supplement fetuses, 57.2 against 78.1 pmol O₂/min/µg protein in control + supplement.
• Liver mtDNA copy number went up with restricted gain and with supplementation. In muscle, only restricted gain increased it.
• Muscle respiration didn't differ between groups at all.
• In muscle, genes such as ACSM1, FABP4, FASN and PCK1/PCK2 were down-regulated in treated groups compared with control fetuses without supplements. RPL12 went the other way and was strongly up-regulated in both tissues.

Discussion

• The liver changed its mitochondrial respiration with no significant changes in mitochondrial gene expression. This study described this as protein-level control, though protein modifications weren't measured directly.
• Muscle showed the opposite pattern, with changes in gene expression but no change in function yet. That could be programming that shows up after birth, in form of feed efficiency or growth.
• An earlier look at the same treatments at day 63 found no respiration effect, so the fetal liver seems to become more responsive as it matures.
• Gut development in fetuses from this trial is covered in our Veterinary Sciences papers also available at the research page. For a genetics view of heifer growth, see the beef heifer GWAS case study.
• All heifers retained had female fetuses only, and all dams were first-calf heifers, so results may differ for male calves or older cows with different parity.

Conclusion

• Maternal diet in the first 63 days of pregnancy still shaped fetal mitochondria at day 161.
• Fetal liver responded with more respiration and mtDNA. Fetal muscle responded through its gene expression regulation instead.
• Whether these changes last to birth and affect calf performance is still an open question.

Key References

1. Daneshi M, Borowicz PP, Entzie YL, Syring JG, King LE, Safain KS, Anas M, Reynolds LP, Ward AK, Dahlen CR, Crouse MS, Caton JS (2024). Influence of maternal nutrition and one-carbon metabolites supplementation during early pregnancy on bovine fetal small intestine vascularity and cell proliferation. Veterinary Sciences, 11, 146. https://doi.org/10.3390/vetsci11040146
2. Bazer FW, Spencer TE, Wu G, Cudd TA, Meininger CJ (2004). Maternal nutrition and fetal development. Journal of Nutrition, 134, 2169–2172. https://doi.org/10.1093/jn/134.9.2169
3. Du M, Tong J, Zhao J, Underwood KR, Zhu M, Ford SP, Nathanielsz PW (2010). Fetal programming of skeletal muscle development in ruminant animals. Journal of Animal Science, 88 (Suppl. 13), E51–E60. https://doi.org/10.2527/jas.2009-2311
4. Crouse MS, Caton JS, Cushman RA, McLean KJ, Dahlen CR, Borowicz PP, Reynolds LP, Ward AK (2019). Moderate nutrient restriction of beef heifers alters expression of genes associated with tissue metabolism, accretion, and function in fetal liver, muscle, and cerebrum by day 50 of gestation. Translational Animal Science, 3, 855–866. https://doi.org/10.1093/tas/txz026
5. Syring JG, Crouse MS, Entzie YL, King LE, Hirchert MR, Ward AK, Reynolds LP, Borowicz PP, Dahlen CR, Caton JS (2024). One-carbon metabolite supplementation increases vitamin B12, folate, and methionine cycle metabolites in beef heifers and fetuses in an energy dependent manner at day 63 of gestation. Journal of Animal Science, 102, skae202. https://doi.org/10.1093/jas/skae202