Maternal protein supplementation during mid-gestation improves offspring performance and metabolism in beef cows.

This study examined the impact of maternal protein supplementation during mid-gestation on offspring, considering potential sex-related effects. Forty-three pregnant purebred Tabapuã beef cows (20 female and 23 male fetuses) were collectively managed in a pasture until 100 d of gestation. From 100 to 200 d of gestation, they were randomly assigned to the restricted group [(RES) - basal diet (75% corn silage + 25% sugar cane bagasse + mineral mixture); n = 24] or control group [(CON) - same basal diet + based-plant supplement [40% of crude protein, 3.5 g/kg of body weight (BW); n = 19]. From 200 d of gestation until parturition, all cows were equally fed corn silage and mineral mixture. During the cow-calf phase, cows and their calves were maintained in a pasture area. After weaning, calves were individually housed and evaluated during the backgrounding (255 to 320 d), growing 1 (321 to 381 d), and growing 2 (382 to 445 d) phases. Offspring's blood samples were collected at 210 and 445 d of age. Samples of skeletal muscle tissue were collected through biopsies at 7, 30, and 445 d of age. Muscle tissue samples were subjected to reverse-transcription quantitative polymerase chain reaction analysis. Prenatal treatment and offspring's sex (when pertinent) were considered fixed effects. The significance level was set at 5%. At mid-gestation, cows supplemented with protein reached 98% and 92% of their protein and energy requirements, while nonsupplemented cows attained only 30% and 50% of these requirements, respectively. The RES offspring were lighter at birth (27 vs. 31 kg), weaning (197 vs. 214 kg), and 445 d of age (398 vs. 429 kg) (P ≤ 0.05). The CON calves had greater (P < 0.05) morphometric measurements overall. The CON offspring had ~26% greater muscle fiber area (P ≤ 0.01). There was a trend (P = 0.06) for a greater Mechanistic target of rapamycin kinase mRNA expression in the Longissimus thoracis in the CON group at 7 d of age. The Myogenic differentiation 1 expression was greater (P = 0.02) in RES-females. Upregulation of Carnitine palmitoyltransferase 2 was observed in RES offspring at 445 d (P = 0.04). Expression of Fatty acid binding protein 4 (P < 0.001), Peroxisome proliferator-activated receptor gamma (P < 0.001), and Stearoyl-Coenzyme A desaturase (P < 0.001) was upregulated in CON-females. Therefore, protein supplementation during gestation enhances offspring growth and promotes favorable responses to lipogenesis, particularly in females.

In tropical conditions, beef cows on pasture often experience protein restriction during mid-to-late gestation, potentially impacting offspring development negatively. To address this, we investigated the effects of strategic protein supplementation for pregnant beef cows fed low-quality forage during mid-gestation on the postnatal growth trajectory of their offspring. The supplementation program, implemented during mid-gestation, increased dry matter intake by addressing nitrogen deficiency in the rumen, resulting in meeting 98% and 92% of protein and energy requirements in supplemented cows. In contrast, nonsupplemented cows met only 30% and 50% of these requirements, respectively. Consequently, protein supplementation positively influenced the postnatal growth trajectory of the offspring, attributed to beneficial changes in secondary myogenesis and hypertrophy processes. Supplementing cows with crude protein also stimulated lipogenesis, potentially contributing to intramuscular fat deposition, particularly in females. Therefore, this study emphasizes the importance of nutritional interventions for pregnant beef cows fed low-quality forage.

Do carcass traits influence consumer perception of pork eating quality?

The objective of this study was to assess carcass traits' influence on pork eating quality as evaluated by consumers. A total of 1360 pork chops were used, with 824 from the sirloin end and 536 from the butt end of the loin (Longissimuss thoracis et lumborum), to produce 340 packages, each containing four pork chops. Untrained participants received one package of either sirloin or butt chops, being two pork chops from barrows and two from gilts. Participants answered a survey rating the tenderness, juiciness, flavour, and overall acceptability of each chop on an 8-point scale. Correlation analysis was conducted between carcass traits and pork eating quality attributes. For the descriptive analysis, classes (low, medium, and high) for carcass traits, Warner-Bratzler shear force (WBSF) and cooking loss were created based on our consumer responses dataset for palatability attributes. No significant correlations (P > 0.05) were observed between carcass traits and pork eating quality traits. Tenderness and overall acceptability were negatively correlated (P < 0.05) with cooking loss and WBSF. Loin intramuscular fat (IMF) content showed a weak negative correlation (P < 0.05) with WBSF and cooking loss. Consumers rated chops from the high and medium/high backfat thickness and loin IMF classes slightly higher for tenderness and juiciness, respectively. Additionally, chops from the low and/or medium WBSF and cooking loss classes received slightly higher scores for tenderness and juiciness than pork chops in the high classes. In conclusion, the study indicated that carcass traits had minimal impact on overall acceptability of pork by consumers.

Can the post-ruminal urea release impact liver metabolism, and nutritional status of beef cows at late gestation?

We aimed to evaluate the effects of post-ruminal supply of urea (PRU) on nutritional status, and liver metabolism of pregnant beef cows during late gestation. Twenty-four Brahman dams, pregnant from a single sire, and weighing 545 kg ± 23 kg were confined into individual pens at 174 ± 23 d of gestation, and randomly assigned into one of two dietary treatments up to 270 d of gestation: Control (CON, n = 12), consisting of a basal diet supplemented with conventional urea, where the cows were fed with diets containing 13.5 g conventional urea per kg dry matter; and PRU (PRU, n = 12), consisting of a basal diet supplemented with a urea coated to extensively prevent ruminal degradation while being intestinally digestible, where the cows were fed with diets containing 14,8 g urea protected from ruminal degradation per kg dry matter. Post-ruminal supply of urea reduced the urine levels of 3-methylhistidine (P = 0.02). There were no differences between treatments for dry matter intake (DMI; P = 0.76), total digestible nutrient (TDN) intake (P = 0.30), and in the body composition variables, such as, subcutaneous fat thickness (SFT; P = 0.72), and rib eye area (REA; P = 0.85). In addition, there were no differences between treatments for serum levels of glucose (P = 0.87), and serum levels of glucogenic (P = 0.28), ketogenic (P = 0.72), glucogenic, and ketogenic (P = 0.45) amino acids, neither for urea in urine (P = 0.51) as well as urea serum (P = 0.30). One the other hand, enriched pathways were differentiated related to carbohydrate digestion, and absorption, glycolysis, pyruvate metabolism, oxidative phosphorylation, pentose phosphate pathway, and biosynthesis of amino acids of the exclusively expressed proteins in PRU cows. Shifting urea supply from the rumen to post-ruminal compartments decreases muscle catabolism in cows during late gestation. Our findings indicate that post-ruminal urea supplementation for beef cows at late gestation may improve the energy metabolism to support maternal demands. In addition, the post-ruminal urea release seems to be able to trigger pathways to counterbalance the oxidative stress associated to the increase liver metabolic rate.

A comparison of carcass characteristics, carcass cutting yields, and meat quality of barrows and gilts.

Objectives of this research were to compare carcass characteristics, carcass cutting yields, and meat quality for market barrows and market gilts. Commercially-sourced carcasses from 168 market barrows and 175 market gilts weighing an average of 107.44 ± 7.37 kg were selected from 17 different slaughter groups representing approximately 3,950 carcasses. Each group was sorted into percentiles based on hot carcass weight with an equal number of barrows and gilts selected from each quartile so that weight minimally confounded parameters of interest. Carcass lean yield was determined for carcasses following fabrication (i.e. dissection of lean, fat, and bone tissue components) and meat quality measurements were evaluated at the time of fabrication (24 to 72 h postmortem) and following 14-d of postmortem storage. Data were analyzed as a randomized complete block design with carcass serving as the experimental unit, sex (barrow or gilt), the three hot carcass weight quantiles (light [<104 kg]; average [104 to 110 kg]; heavy [>110 kg]), and the interaction between sex and hot carcass weight quantile serving as fixed effects, and producer nested within slaughter event serving as a random effect. Results from the study demonstrated that gilt carcasses were leaner (3 mm less backfat thickness; 3.5 cm2 greater loin muscle area, 1.52% greater merchandized-cut yield, and 2.92% greater dissected carcass lean yield; P < 0.01) than barrow carcasses, while loins from barrows were higher quality (0.43% more intramuscular fat and slightly less shear force; P < 0.01) than loins from gilts. While this study confirms the well-known biological principle that barrow carcasses have greater levels of fat deposition and lower levels of carcass leanness when compared with gilt carcasses, this study provides a much-needed quantification of these differences for the commercial industry that will undoubtedly be useful as new technologies emerge in upcoming years.

Differential average daily gain of pregnant Holstein × Gyr dairy heifers causes placental adaptations to support fetal growth and development.

This study aimed to evaluate the effects of differential average daily gain targets of dairy heifers throughout gestation on placental hemodynamics, uterine involution, colostrum production of the heifers, and effects on newborn calf weight and immunity transfer. Fourteen Holstein × Gyr heifers with an average body weight of 446 ± 46.7 kg and age of 25 ± 3.9 mo were randomly assigned to the following treatments: moderate body weight gain (MOD, n = 7), where heifers were fed to achieve 0.50 kg/d; and high body weight gain (HIG, n = 7), where heifers were fed to achieve 0.75 kg/d. Target average daily gains were established based on common tropical dairy production systems. The heifers received a total mixed ration feed twice daily starting at 70 d of gestation. Placentome vascularization was assessed using a color Doppler ultrasound at 180, 210, and 240 d of gestation. After calving, cotyledons were counted and sampled to analyze the mRNA expression of placental angiogenesis markers. After birth, calves were weighed and fed colostrum, and transfer of passive immunity efficiency was assessed. A significant increase in cotyledons was detected for MOD placenta soon after expulsion (81.5 ± 12.91 vs. 63.6 ± 10.52). Placentome vascularization at the final third of gestation increased for MOD heifers compared with HIG. Greater mRNA expression after membrane expulsion of VEGFB and IGFR1 in cotyledons and a greater estradiol concentration in circulation 1 d before calving was found for MOD heifers compared with HIG heifers; however, uterine involution postpartum was not different between treatment groups. Greater colostrum production was observed in HIG heifers (3.9 ± 1.05 vs. 2.2 ± 1.57 L) but with lower quality (25.2 ± 0.51 vs. 29.5 ± 0.65 Brix). No differences were observed in birth weight or transfer of passive immunity efficiency between treatments; however, HIG calves had significantly greater vitality scores than MOD calves. The results of this study indicate that a moderate feeding regimen enhances placental blood flow by increasing angiogenesis, which suggests improved nutrient transfer to the fetus without major effects on its development during the neonatal stage, colostrum production, or uterine involution in the heifers.

Effect of vitamin A injection at birth on intramuscular fat development and meat quality in beef cattle.

This study aimed to evaluate intramuscular fat and expression of genes in the muscle of Montana × Nellore treated with vitamin A at birth. We hypothesized that an injection of vitamin A after birth would increase marbling by increasing the expression of angiogenic, adipogenic, and lipogenic genes. Animals treated with vitamin A had greater marbling in the longissimus muscle (P = 0.05). The vitamin A treatment increased the expression of VEGFA gene at 40 days of age and at weaning and increased the expression of ZNF423 at weaning and at harvesting (P ≤ 0.03). The expression of WNT was higher (P = 0.01) at 40 days of age and at weaning in the animals treated with vitamin A. Vitamin A also increased the expression of SREBF1 at 40 days of age and at weaning (P ≤ 0.05). Therefore, the administration of vitamin A to cattle at birth could be a way to increase carcass marbling without affecting the performance of the animals.

Proteomic analysis reveals changes in energy metabolism of skeletal muscle in beef cattle supplemented with vitamin A.

BACKGROUND: Vitamin A has been reported as a factor influencing marbling deposition in meat from animals. Although the mechanisms by which vitamin A regulates lipid metabolism in mature adipocytes are already well-established, information regarding molecular mechanisms underlying the effects of vitamin A on the regulation of intramuscular fat deposition in beef cattle still remains limited. The present study aimed to assess the molecular mechanisms involved in the intramuscular fat deposition in beef cattle supplemented with vitamin A during the fattening phase using a proteomic approach. RESULTS: Vitamin A supplementation during the fattening phase decreased intramuscular fat deposition in beef cattle. Proteome and phospho-proteome analysis together with biological and networking analysis of the protein differentially abundant between treatments indicated that Vitamin A supplementation affects the overall energy metabolism of skeletal muscle, impairing lipid biosynthesis in skeletal muscle. CONCLUSION: Vitamin A supplementation at fattening phase impairs intramuscular fat deposition in beef cattle likely by changing the energy metabolism of skeletal muscle. The interaction of retinoic acid and heat shock 70-kDa protein may play a pivotal role in intramuscular fat deposition as a consequence of vitamin A supplementation by impairing de novo fatty acid synthesis as a result of a possible decrease in insulin sensitivity in the skeletal muscle. © 2020 Society of Chemical Industry.

Differentially expressed mRNAs, proteins and miRNAs associated to energy metabolism in skeletal muscle of beef cattle identified for low and high residual feed intake.

BACKGROUND: Feed efficiency is one of the most important parameters that affect beef production costs. The energy metabolism of skeletal muscle greatly contributes to variations in feed efficiency. However, information regarding differences in proteins involved in the energy metabolism of the skeletal muscle in beef cattle divergently identified for feed efficiency is scarce. In this study, we aimed to investigate energy metabolism of skeletal muscle of Nellore beef cattle, identified for low and high residual feed intake using a proteomics approach. We further assessed the expression of candidate microRNAs as a one of the possible mechanisms controlling the biosynthesis of the proteins involved in energy metabolism that were differentially abundant between high and low residual feed intake animals. RESULTS: A greater abundance of 14-3-3 protein epsilon (P = 0.01) was observed in skeletal muscle of residual feed intake (RFI) high animals (RFI-High). Conversely, a greater abundance of Heat Shock Protein Beta 1 (P < 0.01) was observed in the skeletal muscle of RFI-Low cattle. A greater mRNA expression of YWHAE, which encodes the 14-3-3 protein epsilon, was also observed in the skeletal muscle of RFI-High animals (P = 0.01). A lower mRNA expression of HSPB1, which encodes the Heat Shock Protein Beta 1, was observed in the skeletal muscle of RFI-High animals (P = 0.01). The miR-665 was identified as a potential regulator of the 14-3-3 protein epsilon, and its expression was greater in RFI-Low animals (P < .001). A greater expression of miR-34a (P = 0.01) and miR-2899 (P < .001) was observed in the skeletal muscle of RFI-High animals, as both miRNAs were identified as potential regulators of HSPB1 expression. CONCLUSION: Our results show that Nellore cattle divergently identified for feed efficiency by RFI present changes in the abundance of proteins involved in energy expenditure in skeletal muscle. Moreover, our data point towards that miR-665, miR34a and miR-2899 are likely involved in controlling both 14-3-3 epsilon and HSPB1 proteins identified as differentially abundant in the skeletal muscle of RFI-High and RFI-Low Nellore cattle.

Effect of maternal feed restriction in dairy goats at different stages of gestation on skeletal muscle development and energy metabolism of kids at the time of births.

The aim was to determine effects of maternal feed restriction in dairy goats at gestational different stages on skeletal muscle development and energy metabolism in kids at birth. Six pregnant goats were fed 50% of total digestible nutrients (TDN) and crude protein (CP) (NRC, 2007) recommendations in the first half of gestation and then fed to 100% of the recommendations in the second half of gestation (treatment R-M). In the other group, eight pregnant goats were fed 100% of TDN and CP in the first half of gestation and 50% of a restricted diet the second half of gestation (treatment M-R). Birth weight, blood glucose concentration, muscle fiber number, and size of kids at birth were not affected by maternal feed restriction. The mRNA and protein abundance of myogenic, adipogenic and fibrogenic markers were not affected (P > 0.05) by maternal diet. With regard to values for variables in kid energy metabolism, mRNA abundance of the glycolic enzyme HKII was less (P = 0.03) in the M-R group. In conclusion, maternal feed restriction in the first or second half of gestation had no affect mRNA abundance on myogenic, adipogenic, and fibrogenic markers nor were there changes in skeletal muscle mesenchymal stem cell population of kids at the time of birth. There, however, may be detrimental effects on energy metabolism by reducing HKII gene expression in skeletal muscle of dairy goat kids at the time of birth.

Explaining meat quality of bulls and steers by differential proteome and phosphoproteome analysis of skeletal muscle.

The objective of this study was to evaluate the differential proteome and phosphoproteome between bulls and steers during conversion of muscle to meat, as well as after 14 days of aging. Twelve male Nellore (Bos taurus indicus) calves were used, and six calves were randomly selected for surgical castration. Calves were fed the same diet and were harvested after 230 days on feed. Longissimus muscle was sampled just after stunning (0d postmortem), at deboning (1d postmortem) and after aging (14d postmortem) for differential proteome analysis. Castration upregulated (P < 0.05) the abundance of glycolytic enzymes, while the oxidative phosphorylation protein ATP5B was downregulated (P < 0.05). In addition, abundance of troponin T fast isoform (TNNT3) was upregulated by castration (P < 0.05), while the slow isoform (TNNT1) tended to be decreased (P < 0.10). The creatine kinase M-type was markedly fragmented postmortem. Abundance of phosphorylated PGM1 increased during the first 24 h postmortem and was highly correlated with carcass pH. Further, abundance of the phosphorylated myofibrillar proteins ACTA1 and MYLPF were positively correlated with sarcomere shortening. Overall, our finds demonstrated that abundance and phosphorylation of glycolytic enzymes are associated with changes in beef tenderness and intramuscular fat. SIGNIFICANCE: The design of the present study allowed to clarify the key proteins related to changes during conversion of muscle to meat such as pH decline and sarcomere shortening. In addition, the correlation between some biomarker and meat quality traits were confirmed.

Morphological and molecular differences in corpus luteum of pregnant sows from divergent genetic groups.

Comprehending mechanisms controlling corpus luteum (CL) angiogenesis and apoptosis in pregnant sows is essential to understand the physiological role of these processes in CL function, progesterone production and consequently in conceptus development and prenatal mortality. CL from 54 sows from two genetic groups, a commercial line (COM) and the local Piau breed (LPB), were obtained for gene expression (n = 3 COM; n = 6 LPB), histological and protein analysis (n = 3 COM; n = 3 LPB), divided in six gestational ages (seven, 15, 30, 45, 60 and 90 days). We observed differences between gestational ages in CL morphology, in which the average number of blood vessels/capillaries at 90-days was greater than at the seventh day by Tukey test. RT-qPCR analysis revealed that apoptotic genes (BAX, BCL2 and CASP3) were differentially expressed between genetic groups and gestational ages in each group. Angiogenesis genes also presented differences between genetic groups (ANGPT1) and gestational ages (MMP9, VEGFA and ANGPT1). No differences in protein abundance of steroidogenic enzymes (CYP11A1 and HSD3B1) were observed. Our findings indicate that despite the differences in gene expression, differences in corpus luteum vascularization were observed only across gestational ages, with no dissimilarities between genetic groups.

Molecular Factors Underlying the Deposition of Intramuscular Fat and Collagen in Skeletal Muscle of Nellore and Angus Cattle.

Studies have shown that intramuscular adipogenesis and fibrogenesis may concomitantly occur in skeletal muscle of beef cattle. Thus, we hypothesized that the discrepancy of intramuscular fat content in beef from Nellore and Angus was associated with differences in intramuscular adipogenesis and fibrogenesis during the finishing phase. To test our hypothesis, longissimus muscle samples of Nellore (n = 6; BW = 372.5 ± 37.3 kg) and Angus (n = 6; BW = 382.8 ± 23.9 kg) cattle were collected for analysis of gene and protein expression, and quantification of intramuscular fat and collagen. Least-squares means were estimated for the effect of Breed and differences were considered at P ≤ 0.05. A greater intramuscular fat content was observed in skeletal muscle of Angus compared to Nellore cattle (P≤0.05). No differences were observed for mRNA expression of lipogenic and lipolytic markers ACC, FAS, FABP4, SERBP-1, CPT-2, LPL, and ACOX (P > 0.05) in skeletal muscle of Nellore and Angus cattle. Similarly, no differences were observed in mRNA expression of adipogenic markers Zfp423, PPARγ, and C/EBPα (P>0.05) However, a greater PPARγ protein content was observed in skeletal muscle of Angus compared to Nellore cattle (P≤0.05). A greater abundance of adipo/fibrogenic cells, evaluated by the PDGFRα content, was observed in skeletal muscle of Angus than Nellore cattle (P≤0.05). No differences in fibrogenesis were observed in skeletal muscle of Angus and Nellore cattle, which is in accordance with the lack of differences in intramuscular collagen content in beef from both breeds (P>0.05). These findings demonstrate that difference in intramuscular fat content is associated with a slightly enhanced adipogenesis in skeletal muscle of Angus compared to Nellore cattle, while no difference in fibrogenesis.

Identification of Suitable Reference Genes for Real Time Quantitative Polymerase Chain Reaction Assays on Pectoralis major Muscle in Chicken (Gallus gallus ).

Thirteen reference genes were investigated to determine their stability to be used as a housekeeping in gene expression studies in skeletal muscle of chickens. Five different algorithms were used for ranking of reference genes and results suggested that individual rankings of the genes differed among them. The stability of the expression of reference genes were validated using samples obtained from the Pectoralis major muscle in chicken. Samples were obtained from chickens in different development periods post hatch and under different nutritional diets. For gene expression calculation the ΔΔCt approach was applied to compare relative expression of pairs of genes within each of 52 samples when normalized to mitochondrially encoded cytochrome c oxidase II (MT-CO2) target gene. Our findings showed that hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyl transferase 1 (HPRT1) are the most stable reference genes while transferrin receptor (TFRC) and beta-2-microglobulin (B2M) ranked as the least stable genes in the Pectoralis major muscle of chickens. Moreover, our results revealed that HMBS and HPRT1 gene expression did not change due to dietary variations and thus it is recommended for accurate normalization of RT-qPCR data in chicken Pectoralis major muscle.

Achieving body weight adjustments for feeding status and pregnant or non-pregnant condition in beef cows.

BACKGROUND: Beef cows herd accounts for 70% of the total energy used in the beef production system. However, there are still limited studies regarding improvement of production efficiency in this category, mainly in developing countries and in tropical areas. One of the limiting factors is the difficulty to obtain reliable estimates of weight variation in mature cows. This occurs due to the interaction of weight of maternal tissues with specific physiological stages such as pregnancy. Moreover, variation in gastrointestinal contents due to feeding status in ruminant animals is a major source of error in body weight measurements. OBJECTIVES: Develop approaches to estimate the individual proportion of weight from maternal tissues and from gestation in pregnant cows, adjusting for feeding status and stage of gestation. METHODS AND FINDINGS: Dataset of 49 multiparous non-lactating Nellore cows (32 pregnant and 17 non-pregnant) were used. To establish the relationships between the body weight, depending on the feeding status of pregnant and non-pregnant cows as a function of days of pregnancy, a set of general equations was tested, based on theoretical suppositions. We proposed the concept of pregnant compound (PREG), which represents the weight that is genuinely related to pregnancy. The PREG includes the gravid uterus minus the non-pregnant uterus plus the accretion in udder related to pregnancy. There was no accretion in udder weight up to 238 days of pregnancy. By subtracting the PREG from live weight of a pregnant cow, we obtained estimates of the weight of only maternal tissues in pregnant cows. Non-linear functions were adjusted to estimate the relationship between fasted, non-fasted and empty body weight, for pregnant and non-pregnant cows. CONCLUSIONS: Our results allow for estimating the actual live weight of pregnant cows and their body constituents, and subsequent comparison as a function of days of gestation and feeding status.

Bovine dedifferentiated adipose tissue (DFAT) cells: DFAT cell isolation.

Dedifferentiated fat cells (DFAT cells) are derived from lipid-containing (mature) adipocytes, which possess the ability to symmetrically or asymmetrically proliferate, replicate, and redifferentiate/transdifferentiate. Robust cell isolation and downstream culture methods are needed to isolate large numbers of DFAT cells from any (one) adipose depot in order to establish population dynamics and regulation of the cells within and across laboratories. In order to establish more consistent/repeatable methodology here we report on two different methods to establish viable DFAT cell cultures: both traditional cell culture flasks and non-traditional (flat) cell culture plates were used for ceiling culture establishment. Adipocytes (maternal cells of the DFAT cells) were easier to remove from flat culture plates than flasks and the flat plates also allowed cloning rings to be utilized for cell/cell population isolation. While additional aspects of usage of flat-bottomed cell culture plates may yet need to be optimized by definition of optimum bio-coating to enhance cell attachment, utilization of flat plate approaches will allow more efficient study of the dedifferentiation process or the DFAT progeny cells. To extend our preliminary observations, dedifferentiation of Wagyu intramuscular fat (IMF)-derived mature adipocytes and redifferentiation ability of DFAT cells utilizing the aforementioned isolation protocols were examined in traditional basal media/differentiation induction media (DMI) containing adipogenic inducement reagents. In the absence of treatment approximately 10% isolated Wagyu IMF-mature adipocytes dedifferentiated spontaneously and 70% DFAT cells displayed protracted adipogenesis 12 d after confluence in vitro. Lipid-free intracellular vesicles in the cytoplasm (vesicles possessing an intact membrane but with no any observable or stainable lipid inside) were observed during redifferentiation. One to 30% DFAT cells redifferentiated into lipid-assimilating adipocytes in the DMI media, with distinct lipid-droplets in the cytoplasm and with no observable lipid-free vesicles inside. Moreover, a high confluence level promoted the redifferentiation efficiency of DFAT cells. Wagyu IMF dedifferentiated DFAT cells exhibited unique adipogenesis modes in vitro, revealing a useful cell model for studying adipogenesis and lipid metabolism.

Cellular and molecular implications of mature adipocyte dedifferentiation.

There is a voluminous amount of scientific literature dealing with the involvement of adipocytes in molecular regulation of carcass composition, obesity, metabolic syndrome, or diabetes. To form adipocytes (process termed adipogenesis) nearly all scientific papers refer to the use of preadipocytes, adipofibroblasts, stromal vascular cells or adipogenic cell lines, and their differentiation to form lipid-assimilating cells containing storage triacylglyceride. However, mature adipocytes, themselves, possess ability to undergo dedifferentiation, form proliferative-competent progeny cells (the exact plasticity is unknown) and reinitiate formation of cells capable of lipid metabolism and storage. The progeny cells would make a viable (and alternative) cell system for the evaluation of cell ability to reestablish lipid assimilation, ability to differentially express genes (as compared to other adipogenic cells), and to form other types of cells (multi-lineage potential). Understanding the dedifferentiation process itself and/or dedifferentiated fat cells could contribute to our knowledge of normal growth processes, or to disease function. Indeed, the ability of progeny cells to form other cell types could turn-out to be important for processes of tissue reconstruction/engineering and may have implications in clinical, biochemical or molecular processes.

AMP-activated protein kinase stimulates myostatin expression in C2C12 cells.

AMP-activated protein kinase (AMPK) is a master regulator of energy metabolism in skeletal muscle; AMPK induces muscle protein degradation but the underlying mechanisms are unclear. Myostatin is a powerful negative regulator of skeletal muscle mass and growth in mammalian species. We hypothesized that AMPK stimulates myostatin expression, which provides an explanation for the negative role of AMPK in muscle growth. The objective of this study is to demonstrate that AMPK stimulates myostatin expression using C2C12 cells as a model. Activation of AMPK by 5-aminoimidazole-4-carboxamide-1-ß-d-riboruranoside (AICAR) dramatically increased the mRNA expression and protein content of myostatin in C2C12 myotubes, and to a lesser degree in myoblasts. Metformin, another AMPK activator, also stimulated myostatin expression at low concentrations. In addition, ectopic expression of AMPK wild-type α subunit (enhancing AMPK activity) and AMPK K45R mutant (knockdown AMPK activity) enhanced and reduced myostatin expression, respectively. These results indicate that AMPK stimulates myostatin expression in C2C12 cells, providing an explanation for the negative effect of AMPK on muscle growth.

Like pigs, and unlike other breeds of cattle examined, mature Angus-derived adipocytes may extrude lipid prior to proliferation in vitro.

A large number of studies have shown that mature adipocytes are able to dedifferentiate in vitro into progeny cells, which possess proliferative capacity and mutilineage potential. Our present study confirms that mature adipocytes derived from Angus cattle also dedifferentiate into proliferative-competent progeny cells. However, this report is unlike any published for all other breeds of cattle we have worked with or that we have seen in published reports, in which mature adipocytes retain and distribute lipids into daughter cells symmetrically or asymmetrically. In the present work, we noted that Angus-derived mature adipocytes extruded a majority of their cellular lipid droplets prior to cell division. In this manner, these cells are processing lipid in a manner observed in mature adipocytes isolated from swine tissue. These results suggest that regulation of the mechanism(s) underlying lipid processing might be different between and within animal breeds. Lipid processing in beef-derived adipocytes during dedifferentiation may serve as a unique animal model for studying lipid metabolism during reverse adipogenesis.