Claudia Kalbe

Evidence for estrogen receptor α and β expression in skeletal muscle of pigs

Recent research suggests that estrogen receptors (ERs) are of significance in skeletal muscle function. The aim of the present study was to investigate, whether ERα and ERβ are expressed in different porcine skeletal muscles and in satellite cells derived from semimembranosus muscle (SM) at the protein and mRNA level. Immunohistochemistry demonstrated positive staining for ERα in the nuclei of skeletal muscle cells, while the ERβ stain showed positive signals in nuclei and cytoplasm of skeletal myofibers and myoblasts derived from satellite cells. Additionally, a weak expression of both ER subtypes was seen in skeletal muscle tissue and SM satellite cells with Western blot analysis. A clear expression of the ERα mRNA and a weak expression of the ERβ mRNA was seen in skeletal muscle tissue and SM satellite cell cultures, as determined by reverse transcription (RT)-PCR. The present study shows for the first time that both ERα and ERβ are expressed in porcine skeletal muscle, which, consequently, could be considered as a target tissue for estrogens or estrogen-like compounds. However, more detailed studies on the functional impact of both receptor subtypes in skeletal muscle are necessary. The porcine SM satellite cell culture provides a suitable in vitro model to investigate estrogenic effects on pig skeletal muscle.

Limited and excess dietary protein during gestation affects growth and compositional traits in gilts and impairs offspring fetal growth

The aim of this study was to investigate whether dietary protein intake during gestation less than or greater than recommendations affects gilts growth and body composition, gestation outcome, and colostrum composition. German Landrace gilts were fed gestation diets (13.7 MJ of ME/kg) containing a low (n = 18; LP, 6.5% CP), an adequate (n = 20; AP, 12.1%), or a high (n = 16; HP, 30%) protein content corresponding to a protein:carbohydrate ratio of 1:10.4, 1:5, and 1:1.3, respectively, from mating until farrowing. Gilts were inseminated by semen of pure German Landrace boars and induced to farrow at 114 d postcoitum (dpc; Exp. 1). Energy and protein intake during gestation were 33.3, 34.4, and 35.8 MJ of ME/d (P < 0.001) and 160, 328, and 768 g/d, respectively, in LP, AP, and HP gilts (P < 0.001). From insemination to 109 dpc, BW gain was least in LP (42.1 kg), intermediate in HP (63.1 kg), and greatest in AP gilts (68.3 kg), whereas increase of backfat thickness was least in gilts fed the HP diet compared with LP and AP diets (3.8, 5.1, 5.0 mm; P = 0.01). Litter size, % stillborn piglets, and mummies were unaffected (P > 0.28) by the gestation diet. Total litter weight tended to be less in the offspring of LP and HP gilts (14.67, 13.77 vs. 15.96 kg; P = 0.07), and the percentage of male piglets was greater in litters of HP gilts (59.4%; P < 0.01). In piglets originating from LP and HP gilts, individual birth weight was less (1.20, 1.21 vs. 1.40 kg; P = 0.001) and birth weight/crown-rump length ratio was reduced (45.3, 46.4 vs. 50.7 g/cm; P = 0.003). Colostrum fat (7.8, 7.4 vs. 8.1%) and lactose concentrations (2.2, 2.1 vs. 2.6%) tended to be reduced in LP and HP gilts (P = 0.10). In Exp. 2, 28 gilts (LP, 10; AP, 9; HP, 9) were treated as in Exp. 1 but slaughtered at 64 dpc. At 64 dpc, LP gilts were 7% lighter than AP gilts (P = 0.03), whereas HP gilts were similar to AP gilts. Body composition was markedly altered in response to LP and HP feeding with less lean (P < 0.01) and greater fat content (P = 0.02 to 0.04) in LP and less fat content (P = 0.02 to 0.04) in HP gilts. Fetal litter weight and number, and embryonic survival at 64 dpc were not affected by the diets. These results indicated that gestation diets containing protein at 50 and 250% of recommendations and differing in protein:carbohydrate ratio led to marked changes in protein and fat metabolism in gilts resulting in fetal growth retardation of 15%, which mainly occurred during the second half of gestation.

Effects of limited and excess protein intakes of pregnant gilts on carcass quality and cellular properties of skeletal muscle and subcutaneous adipose tissue in fattening pigs

The aim of this study was to investigate whether dietary protein intake of gilts during gestation below (50%) or above (250%) recommendations affects body composition, carcass and meat quality, and properties of skeletal muscle and subcutaneous adipose tissue (SCAT) in offspring at d 83 and 188 of age. German Landrace gilts were fed isoenergetic gestation diets (~13.7 MJ of ME/kg) containing a low (LP, 6.5%; n = 18), an adequate (AP, 12.1%; n = 20), or a high (HP, 30%; n = 16) protein content from mating until farrowing. Within 48 h of birth, offspring were cross-fostered to sows fed a standard diet. On d 83 of age, no effects of the LP diet on BW and body composition were detected, whereas HP pigs showed a slight growth delay (P = 0.06) associated with increased relative weights of small intestine (P < 0.01) and brain (P = 0.08), and reduced relative thymus weight (P < 0.01). On d 188 of age, BW was not different among the dietary groups. However, the carcass of LP pigs contained less (P = 0.01) lean and more (P = 0.07) fat compared with AP and HP pigs, which was only pronounced in pigs originating from large litters (P < 0.05). Like skeletal muscles (P = 0.06), the heart muscle weighed less (P = 0.02) in LP than AP pigs. Compared with AP pigs, LP pigs exhibited a fewer (P = 0.09) total number of myofibers in semitendinosus muscle plus LM both at d 83 and 188 of age, whereas total muscular DNA was less (P = 0.02) at d 188 only. The mRNA abundance of IGF2 measured on d 188 was reduced in SCAT (P = 0.03) and LM (P = 0.07) of LP compared with AP pigs. No changes in muscular fiber type frequency, capillary density, or creatine kinase activity, as well as SCAT adipocyte size and number, were observed at either stages of age. Meat quality characteristics remained unchanged at d 83, whereas Warner-Bratzler shear force value in LM was decreased (P = 0.03) in LP compared with AP pigs on d 188 of age. The results suggest that the maternal LP diet impairs prenatal myofiber formation, reduces the potential of postnatal lean growth related to reduced IGF2 mRNA expression and myonuclear accumulation, and consequently changes carcass quality toward reduced lean proportion and improved tenderness at market weight. In contrast, except for a slight transient growth delay, excess dietary protein during gestation seems to have little effect on the fetal programming of postnatal muscle and adipose tissue phenotype of the progeny.

Developmental changes and the impact of isoflavones on mRNA expression of IGF-I receptor, EGF receptor and related growth factors in porcine skeletal muscle cell cultures

OBJECTIVE Soy that is widely used in human nutrition and in livestock production is a rich source of isoflavones. In addition to the estrogenic or antiestrogenic effects, isoflavones are suggested to affect cell growth via inhibition of protein tyrosine kinases (e.g. growth factor receptors). Therefore, the present in vitro-study was undertaken to determine, whether genistein and daidzein affect the mRNA expression of growth factor receptors (IGF-I receptor and EGF receptor) and their related growth factors in porcine skeletal muscle cell cultures. DESIGN First, we investigated the basal mRNA expression of IGF-I, IGF-II, EGF, IGF-I receptor, and EGF receptor in proliferating and differentiating porcine skeletal muscle cell cultures using real-time PCR. Secondly, we measured the changes in the mRNA expression in these cell cultures treated with 0 (control), 1, 10, 100 microM genistein or daidzein over 26 h in serum-free medium (n=3). RESULTS The mRNA expression of IGF-I was slightly decreased, whereas transcript concentrations of IGF-II and EGF were increased during differentiation compared with the proliferating stage of porcine muscle cell cultures. IGF-I receptor transcripts tended to be increased, whereas EGF receptor mRNA expression remained unchanged from proliferation to differentiation. Genistein and daidzein at 1 microM and 10 microM showed no effects on the mRNA expression of these genes, neither in proliferating nor in differentiating cells. However, high-concentrated isoflavones (100 microM) decreased the mRNA expression of IGF-I receptor and of the growth factors examined. CONCLUSIONS The present study confirms the role of the IGF and EGF system in proliferation and differentiation of skeletal muscle cell culture especially under serum-free culture conditions. Furthermore, the results of this in vitro-study suggest that there is no effect of isoflavones at concentrations resulting from dietary consumption (1 and 10 microM) on IGF- and EGF-associated gene expression in porcine skeletal muscle tissue. Genistein and daidzein at high concentration (100 microM) reduced the mRNA expression of the IGF-I receptor and the growth factors examined, and therefore, may modify their autocrine and paracrine actions in skeletal muscle tissue.

L-Carnitine supplementation during suckling intensifies the early postnatal skeletal myofiber formation in piglets of low birth weight.

Piglets of low birth weight exhibit a reduced total number of skeletal myofibers at birth and throughout life compared with piglets of middle and heavy birth weight, which is associated with impaired (lean) growth and quality of carcass and meat at market weight. We investigated the effect of L-carnitine supplementation to suckling piglets of different birth weights on early postnatal myofiber formation, muscle growth, and body composition. A total of 48 piglets of low (LW) and middle (MDW) birth weight from 9 German Landrace gilts received 400 mg of L-carnitine (carnitine, n = 25) or a placebo (control, n = 23) once daily from d 7 to 27 of age and were slaughtered on d 28 of age (weaning). Carnitine-supplemented piglets deposited less fat as indicated by a reduced proportion of perirenal (P = 0.1) and intramuscular fat (P = 0.05). Circulating glucose concentrations tended to be greater in supplemented LW piglets (P = 0.13). The concentration of carnitine in semitendinosus (STN) muscle was approximately doubled (P < 0.001) by supplementation, with emphasis on the proportion of esterified carnitine. The ratio of lactate dehydrogenase to isocitrate dehydrogenase tended (P = 0.12) to be smaller in STN muscle of supplemented piglets, indicating a more oxidative muscle metabolism. The total number of STN myofibers was increased by 13% (P = 0.02) in supplemented LW piglets, thereby reaching the unchanged level of MDW littermates. In addition, supplemented LW piglets displayed a 2.4-fold mRNA expression of the gene encoding the embryonic isoform of the myosin heavy chain in STN muscle than control piglets (P = 0.05), but there were no differences in the proportion of fibers positively staining for the embryonic myosin isoform. L-carnitine-supplemented piglets exhibited a greater DNA:protein ratio (P = 0.02) in STN muscle, which resulted from a greater DNA concentration (P = 0.04). However, the STN muscle of L-carnitine-supplemented piglets was not less mature as indicated by unchanged myofiber size, creatine kinase activity, and protein concentration. The results indicate that energy balance has been improved through intensified fatty acid oxidation. As a consequence, myogenic proliferation appears to be stimulated, which in LW piglets may have contributed to a compensatory increase in myofiber number. Thus, piglets, particularly those of low birth weight, could profit from an early postnatal L-carnitine supplementation, which may attenuate the negative consequences of low birth weight on body composition and meat quality at market weight.

Growth performance, carcass characteristics and bioavailability of isoflavones in pigs fed soy bean based diets

A growth trial with 38 weaners (castrated male pigs) was designed to compare the growth performance and carcass quality of pigs fed diets containing either soy bean meal or soy protein concentrate in a pair-feeding design. Soy bean meal (SBM) and soy protein concentrate (SPC) differed in isoflavone (daidzein plus genistein) content (782 μg/g in SBM and 125 μg/g in SPC, respectively). During the experiment, all pigs were fed four-phases-diets characterized by decreasing protein concentrations with increasing age (weaner I, weaner II, grower, finisher diets). Rations of control and experimental groups were isoenergetic, isonitrogenous, and isoaminogen. The weanling pigs with an initial live weight of 8.4 ± 1.1 kg were allotted to flat deck boxes. During the growing/finishing period (days 70 – 170 of age), the pigs were housed in single boxes. Both, the weaning and the grower/finishing performances (daily body weight gain, feed intake, feed conversion ratio) were similar in both groups. No differences were found between the groups in carcass composition (percentages of cuts, tissues, and protein/fat), and meat quality of pigs. Moreover, the IGF-1R mRNA expression in longissimus muscle was not influenced by the kind of soy product. However, circulating levels of isoflavones were clearly different between pigs fed SBM (genistein 239 ± 44; daidzein 162 ± 42; equol 12 ± 4 ng/ml plasma) and animals fed SPC (genistein 22 ± 9 and daidzein 8 ± 3, and equol 10 ± 3 ng/ml plasma). The results confirm the expected differences in the bioavailability of soy isoflavones, yet, there were no significant differences in performance of pigs fed either soy bean meal or soy protein concentrate.

Effects of Dietary Isoflavones on Proliferation and DNA Integrity of Myoblasts Derived from Newborn Piglets

Soy-based formulas are consumed by growing numbers of infants and used as regular food supplements in livestock production. Moreover, constituent dietary phytoestrogens may compete with endogenous estrogens and affect individual growth. This study aimed to investigate the in vitro effects of isoflavones in comparison with estrogens on the proliferation of porcine satellite cells derived from neonatal muscle. After 7 h of exposure in serum-free medium, 17β-estradiol (1 nM, 1 μM), estrone (1 μM), and daidzein (1, 100 μM) slightly decreased whereas 100 μM genistein substantially lowered DNA synthesis. Declines in DNA amount were observed with genistein (1, 100 μM) and daidzein (100 μM). After 26 h of exposure, 100 μM genistein reduced DNA synthesis, whereas it was increased by 10 μM genistein and 10 and 100 μM daidzein. In the case of 10 μM genistein and 100 μM daidzein, these increases apparently resulted from the repair of damaged DNA. Genistein and daidzein (100 μM) reduced protein synthesis, caused a G2/M phase block, and decreased DNA amount in association with higher rates of cell death partially resulting from apoptosis. Conclusively, isoflavones at concentrations of greater than 1 μM act as inhibitors of porcine skeletal muscle cell proliferation.

Dose-dependent effects of genistein and daidzein on protein metabolism in porcine myotube cultures.

This study was conducted to investigate whether the isoflavones genistein and daidzein, which are components of soy-based diets, and the estrogen 17beta-estradiol affect differentiation and protein metabolism of porcine skeletal muscle cells in vitro. Serum-free porcine myotube cultures expressing the estrogen receptors ERalpha and ERbeta were treated with various concentrations of genistein, daidzein, or 17beta-estradiol for 26 h. The degree of differentiation by creatine phosphokinase activity was not altered by treatment. At 100 micromol/L both genistein and daidzein caused decreases in protein amount due to cell loss. In addition, 100 micromol/L genistein reduced protein synthesis rate of the surviving cells (P < 0.05) measured as [3H]-phenylalanine incorporation. Interestingly, genistein (0.1 micromol/L), daidzein (10, 100 micromol/L), and 17beta-estradiol (0.1, 1 nmol/L) slightly reduced protein degradation (P < 0.05). The results suggest that both genistein and daidzein affect protein metabolism in a dose-dependent manner and that estrogenic actions may play a role in decreasing protein degradation in porcine skeletal muscle.

IGF-I- and EGF-dependent DNA synthesis of porcine myoblasts is influenced by the dietary isoflavones genistein and daidzein

Soy-derived isoflavones have been reported to be specific inhibitors of protein tyrosine kinases like the type 1 insulin-like growth factor receptor (IGF-1R) and the epidermal growth factor receptor (EGFR). This study was conducted to investigate, whether IGF-I and EGF stimulate porcine myoblast growth and whether the responses are influenced by isoflavones. Satellite cell-born myoblasts derived from the semimembranosus muscle of newborn piglets were treated for 26 h with IGF-I or EGF alone and in combination with genistein or daidzein. The DNA amount was measured and DNA synthesis was recorded as 6 h-[(3)H]thymidine incorporation during exponential growth in serum-free basal medium. IGF-I and EGF synergistically stimulated DNA synthesis of porcine myoblast with EGF causing a greater response. Genistein (100 micromol/l) effectively reduced the growth factor-mediated DNA synthesis, which was associated with an inhibition of growth factor receptor protein expression. In response to daidzein no reduction in growth factor-mediated DNA synthesis was found. Daidzein (1; 10 micromol/l) combined with IGF-I caused even a slight increase in DNA amount compared with the untreated control. The expression of the IGF-1R precursor protein was reduced with 10 and 100 micromol/l daidzein, whereas the EGFR expression remained unchanged with daidzein. The results suggest that dietary isoflavones may interact with growth factor-induced stimulation of pig skeletal muscle growth.

Predicted high-performing piglets exhibit more and larger skeletal muscle fibers

Postnatal (muscle) growth potential in pigs depends on the total number and hypertrophy of myofibers in skeletal muscle tissue. In a previous study an algorithm was developed to predict piglet BW at the end of the nursery period (10 wk of age) on the basis of BW at birth, at weaning, and at 6 wk of age. The objective of this study was to determine whether the differences in growth performance between poor (PP) and high (HP) performing piglets could be the result of different skeletal muscle properties. Therefore, from a total of 368 piglets (offspring from Hypor sows bred to TOPIGS sires) 2 groups with a divergent growth performance were selected at 6 wk of age: HP (n = 20, predicted BW at 10 wk of age 26.8-30.9 kg) and PP (n = 20, predicted BW at 10 wk of age 16.0-22.9 kg). Piglets were euthanized at 10 wk of age, and samples of the semitendinosus muscle (STN) were collected for histochemistry and gene expression analysis using quantitative PCR (qPCR). At 10 wk of age, realized BW did not differ from predicted BW in either group (P > 0.880). The HP piglets exhibited greater ADG and ADFI from 6 to 10 wk and greater BW at birth and 6 and 10 wk of age (P ≤ 0.002) compared with the PP piglets, whereas G:F ratio was similar (P = 0.417). Superior growth performance of HP piglets was associated with a 1.27-fold higher IGF1 plasma concentration at 10 wk compared with the PP piglets (P = 0.044). The greater weight and muscle cross-sectional area of STN in HP piglets was due to a 1.20-fold increase in total muscle fiber number (TFN; P = 0.009) and 1.34-fold increase in fiber cross-sectional area (FCSA; P = 0.004) compared with the PP piglets. The number of myonuclei per red and intermediate fiber was greater in HP piglets (P ≤ 0.097), but the nucleus-to-cytoplasm ratio was unaffected by the performance group (P = 0.861). The mRNA expression of proliferating cell nuclear antigen (PCNA), paired box 7 (PAX7), myogenic factor 5 (MYF5), and myogenic differentiation factor (MYOD) did not differ between groups (P ≥ 0.327). However, IGF2-specific mRNA expression was numerically higher in the HP piglets (P = 0.101). The greater myofiber number, the higher degree of myofiber hypertrophy, and the increased muscular mRNA expression of IGF2 indicate that HP piglets exhibit a greater capacity for lean accretion and may grow faster until market weight. In summary, pigs that were selected for predicted high BW at 10 wk of age using a complex selection model had a superior muscularity in terms of greater TFN and FCSA, which may be of advantage for lean mass accretion in later life and for meat quality.