The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at −80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies.

Early lactation is an energy-demanding period for dairy cows which may lead to negative energy balance, threatening animal health and consequently productivity. Herein we studied hepatic mitochondrial function in Holstein-Friesian multiparous dairy cows during lactation, under two different feeding strategies. During the first 180 days postpartum the cows were fed a total mixed ration (70% forage: 30% concentrate) ad libitum (non-grazing group, G0) or grazed Festuca arundinacea or Mendicago sativa plus supplementation (grazing group, G1). From 180 to 250 days postpartum, all cows grazed Festuca arundinacea and were supplemented with total mixed ration. Mitochondrial function was assessed measuring oxygen consumption rate in liver biopsies and revealed that maximum respiratory rate decreased significantly in grazing cows during early lactation, yet was unchanged in non-grazing cows during the lactation curve. While no differences could be found in mitochondrial content or oxidative stress markers, a significant increase in protein lysine acetylation was found in grazing cows during early lactation but not in cows from the non-grazing group. Mitochondrial acetylation positively correlated with liver triglycerides and β-hydroxybutyrate plasma levels, well-known markers of negative energy balance, while a negative correlation was found with the maximum respiratory rate and sirtuin 3 levels. To our knowledge this is the first report of mitochondrial function in liver biopsies of dairy cows during lactation. On the whole our results indicate that mitochondrial function is impaired during early lactation in grazing cows and that acetylation may account for changes in mitochondrial function in this period. Additionally, our results suggest that feeding total mixed ration during early lactation may be an efficient protective strategy.

In this study, we explored mechanisms related to glucose and fatty acid metabolism in Holstein–Friesian multiparous dairy cows during lactation under two feeding strategies. From 0 to 180 days postpartum, cows were fed total mixed ration (TMR) ad libitum (non-grazing group, G0) or grazed Festuca arundinacea or Medicago sativa and were supplemented with 5.4 kg DM/d of an energy-protein concentrate (grazing group, G1). From 180 to 250 days postpartum, all cows grazed F. arundinacea and were supplemented with TMR. Plasma samples and liver biopsies were collected at −14, 35, 60, 110, 180, and 250 days in milk (DIM) for metabolite, hormone, gene expression, and western blot analysis. Our results showed increased levels of negative energy balance markers: plasma non-esterified fatty acids (NEFA), liver triglyceride and plasma β-hydroxybutyrate (BHB) (P < 0.01), triglyceride and β-hydroxybutyrate concentration were especially elevated for G1 cows. Also, hepatic mRNA expression of gluconeogenic enzymes was upregulated during early lactation (P < 0.05). In particular, methymalonyl-CoA mutase expression was increased for G0 cows (P < 0.05) while pyruvate carboxylase (PC) expression was increased for G1 cows (P < 0.05), suggesting differential gluconeogenic precursors for different feeding strategies. Phosphorylation of AMP-activated protein kinase was increased in early lactation vs. late lactation (P < 0.01) and negatively correlated with PC mRNA levels. The positive association of gluconeogenic genes with proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) hepatic expression supported the importance of this transcription factor in glucose metabolism. The peroxisome proliferator-activated receptor alpha (PPARA) mRNA was increased during early lactation (P < 0.05), and was positively associated to PPARGC1A, carnitine palmitoyl-transferase 1, and hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) mRNA expression. Alongside, hepatic mRNA expression of FABP was decreased for G1 vs. G0 cows (P < 0.05), possibly linked to impaired fatty acid transport and related to accumulation of liver triglycerides, evidencing G1 cows fail to adapt to the demands of early lactation. In sum, our results showed that metabolic adaptations related to early lactation negative energy balance can be affected by feeding strategy and might be regulated by the metabolic sensors AMPK, SIRT1, and coordinated by transcription factors PPARGC1A and PPARA.

No abstract

Oxidative stress occurs when oxidant production exceeds the antioxidant capacity to detoxify the reactive intermediates or to repair the resulting damage. Feed efficiency has been associated with mitochondrial function due to its impact on cell energy metabolism. However, mitochondria are also recognized as a major source of oxidants. The aim of this study was to determine lipid and protein oxidative stress markers, and gene and protein expression as well as activity of antioxidant enzymes in the liver of steers of divergent residual feed intake (RFI) phenotypes. Hereford steers (n = 111) were evaluated in post-weaning 70 days standard test for RFI. Eighteen steers exhibiting the greatest (n = 9; high-RFI) and the lowest (n = 9; low-RFI) RFI values were selected for this study. After the test, steers were managed together under grazing conditions until slaughter when they reached the slaughter body weight. At slaughter, hepatic samples were obtained, were snap-frozen in liquid nitrogen and stored at −80°C until analyses. Hepatic thiobarbituric acid reactive species and protein carbonyls were greater (P = 0.05) and hepatic 4-hydroxynonenal protein adducts tended (P = 0.10) to be greater for high- than low-RFI steers. Hepatic gene expression glutathione peroxidase 4, glutamate–cysteine ligase catalytic subunit and peroxiredoxin 5 mRNA was greater (P ≤ 0.05) and glutathione peroxidase 3 mRNA tended (P = 0.10) to be greater in low- than high-RFI steers. Hepatic protein expression and enzyme activity of manganese superoxide dismutase and glutathione peroxidase enzyme activity tended (P ≤ 0.10) to be greater for low- than high-RFI steers. High-efficiency steers (low-RFI) probably had better hepatic oxidative status which was strongly associated with greater antioxidant ability near to the oxidant production site and, therefore, reduced oxidative stress of the liver. Decreased hepatic oxidative stress would reduce maintenance requirements due to a lower protein and lipid turnover and better efficiency in the use of energy.

Variations in phenotypic expression of feed efficiency could be associated with differences or inefficiencies in mitochondria function due to its impact on energy expenditure. The aim of this study was to determine hepatic mitochondrial density and function in terms of respiration, gene and protein expression, and enzyme activity of mitochondrial respiratory complex proteins, in steers of divergent residual feed intake (RFI) phenotypes. Hereford steers (n = 111 and n = 122 for year 1 and 2, respectively) were evaluated in postweaning 70 d standard test for RFI. Forty-six steers exhibiting the greatest (n = 9 and 16 for year 1 and 2; high-RFI) and the lowest (n = 9 and 12 for year 1 and 2; low-RFI) RFI values were selected for this study. After the test, steers were managed together until slaughter under grazing conditions until they reached the slaughter body weight. At slaughter, hepatic samples (biopsies) were obtained. Tissue respiration was evaluated using high-resolution respirometry methods. Data were analyzed using a mixed model that included RFI group as fixed effect and slaughter date and year as a random effect using PROC MIXED of SAS. RFI and dry matter intake were different (P < 0.001) between low and high-RFI groups of year 1 and year 2. Basal respiration and maximum respiratory rate were greater (P ≤ 0.04) for low than high-RFI steers when complex II substrates (succinate) were supplied. However, when Complex I substrates (glutamate/malate) were used maximum respiratory capacity tended to be greater (P < 0.09) for low vs. high-RFI steers. Low-RFI steers presented greater mitochondria density markers (greater (P < 0.05) citrate synthase (CS) activity and tended (P ≤ 0.08) to have greater CS mRNA and mtDNA:nDNA ratio) than high-RFI steers. Hepatic expression SDHA, UQCRC1, and CYC1 mRNA was greater (P ≤ 0.02) and expression of NDUFA4, NDUFA13, SDHD, UQCRH, and ATP5E mRNA tended (P ≤ 0.10) to be greater in low than high-RFI steers. Hepatic SDHA protein expression tended (P < 0.08) to be greater while succinate dehydrogenase activity was greater (P = 0.04) and NADH dehydrogenase activity was greater (P = 0.03) for low than high-RFI steers. High-efficiency steers (low-RFI) probably had greater efficiency in hepatic nutrient metabolism, which was strongly associated with greater hepatic mitochondrial density and functioning, mainly of mitochondrial complex II.

The objective of this study was to assess hepatic ATP synthesis in Holstein cows of North American and New Zealand origins and the gluconeogenic pathway, one of the pathways with the highest ATP demands in the ruminant liver. Autumn-calving Holstein cows of New Zealand and North American origins were managed in a pasture-based system with supplementation of concentrate that represented approximately 33% of the predicted dry matter intake during 2017, 2018, and 2019, and hepatic biopsies were taken during mid-lactation at 174 ± 23 days in milk. Cows of both strains produced similar levels of solids-corrected milk, and no differences in body condition score were found. Plasma glucose concentrations were higher for cows of New Zealand versus North American origin. Hepatic mitochondrial function evaluated measuring oxygen consumption rates showed that mitochondrial parameters related to ATP synthesis and maximum respiratory rate were increased for cows of New Zealand compared with North American origin. However, hepatic gene expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate dehydrogenase kinase was increased in North American compared with New Zealand cows. These results altogether suggest an increased activity of the tricarboxylic cycle in New Zealand cows, leading to increased ATP synthesis, whereas North American cows pull tricarboxylic cycle intermediates toward gluconeogenesis. The fact that this occurs during mid-lactation could account for the increased persistency of North American cows, especially in a pasture-based system. In addition, we observed an augmented mitochondrial density in New Zealand cows, which could be related to feed efficiency mechanisms. In sum, our results contribute to the elucidation of hepatic molecular mechanisms in dairy cows in production systems with higher inclusion of pastures.

The integration of feeding behaviour with hepatic and endocrine–metabolic signals provides insights for a better understanding of short-term intake in dairy pasture-based systems. Therefore, the objective was to quantify hepatic and endocrine–metabolic signals before and after the first daily feeding event relating to feeding behaviour in a total mixed ration (TMR) versus a grazing pasture-based diet. During 15 days of adaptation and 5 days of measurements, 14 multiparous Holstein cows (days in milk = 148 ± 12.7; liveweight = 535 ± 10.9 kg; body condition score = 2.8 ± 0.08 (1–5 scale); milk yield = 28.9 ± 3.32 kg) were assigned to two treatments in a randomised block design: PAS = pasture (herbage allowance = 45 kgDM/cow.day; dry matter (DM) = 21%, net energy requirements for maintenance and lactation = 6.7 MJ/kgDM) + concentrate (0.9% of liveweight) or TMR (55:45 forage:concentrate ratio, as-dry basis; DM = 40%, net energy requirements for maintenance and lactation = 7.2 MJ/kgDM) ad libitum in a free stall facility. The DM intake of the first feeding event, feeding behaviour, and total DM intake and milk production, were measured. Blood and liver samples were taken before and after the first feeding event for hormones and metabolites determination. Comparing TMR versus PAS cows, total DM and net energy requirements for maintenance and lactation intake, milk production, and energy balance were greater (P &lt; 0.05), eating and rumination activities were lower (9.2%, P &lt; 0.01; 2.4%, P = 0.06 respectively) and resting activity was greater (11.6%, P &lt; 0.01), whereas duration and DM intake of the first feeding event did not differ. The insulin:glucagon ratio and liver adenosine triphosphate:adenosine diphosphate ratio increased (P &lt; 0.05), and plasma glucose decreased (P &lt; 0.05) after the first feeding event only in TMR cows, probably due to greater flux of propionate to the liver. A negative correlation between post-feeding liver adenosine triphosphate:adenosine diphosphate ratio and post-feeding liver acetyl coenzyme A (r = –0.82, P = 0.045) was also observed only in TMR cows. It is concluded that hepatic and metabolic signals known to support the hepatic oxidation theory in TMR-fed cows appear not to affect the cessation of the first feeding event in mid-lactation cows grazing a pasture-based diet. Further research is required to relate intake rate, flux of nutrients to liver and its response in hepatic metabolism in grazing dairy cows.