Male Reproductive Organ Weight: Criteria for Detection of Androstenone-Positive Carcasses in Immunocastrated and Entire Male Pigs.

Immunocastration and rearing of entire males (EMs) are sustainable alternatives to surgical castration. However, these animal carcasses have variable risk of boar taint and should be identified at the slaughter line. We aimed to identify a simple and reliable indicator of androstenone-related boar taint by evaluating pelvic urogenital tract weight as a marker of boar-taint animals at the slaughter line. The pelvic urogenital tract, testes, and accessory sex glands of EMs and immunocastrates (ICs) were collected, dissected, and weighed, before colorimetric measurements of testicular tissue. Additionally, GnRH antibody titers and testosterone, androstenone, and skatole levels were determined. Our results showed that 81.8% of EMs had androstenone levels above the risk threshold (>0.5 µg/g fat; EM/Ahigh subgroup), whereas in ICs, the C/Ahigh subgroup with androstenone >0.5 µg/g fat accounted for only 4.3%. Androstenone levels correlated negatively with GnRH antibody titers and positively with testosterone levels and reproductive organ weights. Identification of ICs with androstenone levels above the threshold (IC/Ahigh subgroup) may be achieved via testes or pelvic urogenital tract weight measurements. However, in EMs, the latter is a more reliable parameter. A principal component analysis based on these variables and hierarchical clustering also distinguished the Ahigh from the Alow subgroup, irrespective of IC/EM. The findings highlight the possible use of pelvic urogenital tract weight along with testes weight as a simple, reliable, and efficient morphometric indicator for identifying androstenone-positive carcasses of different sex categories.

Salivary Gland Adaptation to Dietary Inclusion of Hydrolysable Tannins in Boars.

The ingestion of hydrolysable tannins as a potential nutrient to reduce boar odor in entire males results in the significant enlargement of parotid glands (parotidomegaly). The objective of this study was to characterize the effects of different levels of hydrolysable tannins in the diet of fattening boars (n = 24) on salivary gland morphology and proline-rich protein (PRP) expression at the histological level. Four treatment groups of pigs (n = 6 per group) were fed either a control (T0) or experimental diet, where the T0 diet was supplemented with 1% (T1), 2% (T2), or 3% (T3) of the hydrolysable tannin-rich extract Farmatan®. After slaughter, the parotid and mandibular glands of the experimental pigs were harvested and dissected for staining using Goldner's Trichrome method, and immunohistochemical studies with antibodies against PRPs. Morphometric analysis was performed on microtome sections of both salivary glands, to measure the acinar area, the lobular area, the area of the secretory ductal cells, and the sizes of glandular cells and their nuclei. Histological assessment revealed that significant parotidomegaly was only present in the T3 group, based on the presence of larger glandular lobules, acinar areas, and their higher nucleus to cytoplasm ratio. The immunohistochemical method, supported by color intensity measurements, indicated significant increases in basic PRPs (PRB2) in the T3 and acidic PRPs (PRH1/2) in the T1 groups. Tannin supplementation did not affect the histo-morphological properties of the mandibular gland. This study confirms that pigs can adapt to a tannin-rich diet by making structural changes in their parotid salivary gland, indicating its higher functional activity.

Immunocastration in adult boars as a model for late-onset hypogonadism.

BACKGROUND: While immunocastration has been studied in male pre-pubertal pigs, data on older, sexually mature animals are limited. To understand the physiological effects of androgen deprivation in the late sexual development phase, we compared mature immunocastrated boars (n = 19; average age = 480 days) to young male immunocastrated pigs (n = 6; average age = 183 days) and young entire males (n = 6; average age = 186 days) as positive and negative controls, respectively. OBJECTIVES: We hypothesized that the timing of gonadotropin-releasing hormone suppression (early or late sexual development phases) influences the extent of reproductive function inhibition, histological structure of testicular tissue, and expression levels of selected genes related to steroid metabolism. MATERIALS AND METHODS: Antibody titer, hormonal status, and histomorphometric analysis of testicular tissue were subjected to principal component analysis followed by hierarchical clustering to evaluate the immunocastration effectiveness in mature boars. RESULTS: Hierarchical clustering differentiated mature immunocastrated boars clustered with young immunocastrated pigs from those clustered with entire males. Although all mature immunocastrated boars responded to vaccination, as evidenced by the increased gonadotropin-releasing hormone antibody titers (p < 0.001), decreased serum luteinizing hormone concentrations (p = 0.002), and changes in testicular tissue vascularization (lighter and less red testicular parenchyma; p ≤ 0.001), the responses were variable. Sharp decreases in testes index (p < 0.001), Leydig cell volume density (p < 0.001), Leydig cell nucleus-to-cytoplasm ratio (p < 0.001), and testosterone concentration (p < 0.001) were observed in mature immunocastrated boars clustered with young immunocastrated pigs compared with those that clustered with entire males. Additionally, mature immunocastrated boars clustered with young immunocastrated pigs showed lower hydroxysteroid 17-beta dehydrogenase 7 expression than entire males (p < 0.05). The young immunocastrated pigs group showed higher follicle-stimulating hormone receptors than the entire males and mature immunocastrated boars, lower steroidogenic acute regulatory protein expression levels compared with entire males, and mature immunocastrated boars clustered with entire males (p < 0.01). CONCLUSION: The two-dose vaccination regime resulted in progressive but variable regression of testicular function in adult (post-pubertal) pigs; however, it was insufficient to induce a complete immunocastration response in all animals.

Adipose Tissue Gene Expression of Entire Male, Immunocastrated and Surgically Castrated Pigs.

Differences in adipose tissue deposition and properties between pig male sex categories, i.e., entire males (EM), immunocastrates (IC) and surgical castrates (SC) are relatively well-characterized, whereas the underlying molecular mechanisms are still not fully understood. To gain knowledge about the genetic regulation of the differences in adipose tissue deposition, two different approaches were used: RNA-sequencing and candidate gene expression by quantitative PCR. A total of 83 differentially expressed genes were identified between EM and IC, 15 between IC and SC and 48 between EM and SC by RNA-sequencing of the subcutaneous adipose tissue. Comparing EM with IC or SC, upregulated genes related to extracellular matrix dynamics and adipogenesis, and downregulated genes involved in the control of lipid and carbohydrate metabolism were detected. Differential gene expression generally indicated high similarity between IC and SC as opposed to EM, except for several heat shock protein genes that were upregulated in EM and IC compared with SC. The candidate gene expression approach showed that genes involved in lipogenesis were downregulated in EM compared with IC pigs, further confirming RNA-sequencing results.

Bone tissue morphology of rat offspring lactationally exposed to polychlorinated biphenyl 169 and 155.

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent, organic pollutants also considered endocrine-disrupting chemicals. Our study examined the effects of lactational exposure to nondioxin-like PCB-155 and/or dioxin-like PCB-169 on longitudinal femur growth at the distal epiphyseal growth plate (EGP) in young rats at three different ages [postnatal days (PNDs) 9, 22, and 42]. After delivery, lactating rats were divided into four groups (PCB-169, PCB-155, PCB-155 + 169, and control) and administered PCBs intraperitoneally. The femurs of offspring were used to estimate growth rate (µm/day), and histomorphometric analysis on the distal femur included the thickness of the EGP and zones of proliferation and hypertrophy with calcification. Stereometry was used to determine trabecular bone volume density. In the PCB-169 and PCB-155 + 169 groups, PCB-169 affected longitudinal bone growth in the early postnatal period by interfering with chondrocytes in the EGP zone of proliferation and, to a lesser extent, the zone of hypertrophy. Morphometric alterations in EGP structure diminished until puberty. A slow growth rate persisted in the PCB-169 group until PND 42, while in the PCB-155 group, a fast growth rate between PNDs 9 to 22 was significantly reduced between PNDs 22 to 42. Sterometric assessment showed decreased trabecular bone volume in the PCB-155 + 169 group compared with that in the control on PND 9 and increased in the PCB-169 group compared with that in the PCB-155 group on PND 42. To summarize, studied PCB congeners exerted congener- and age-dependent effects on femur growth rate and its histomorphometric characteristics.

Expression of Myosin Heavy Chain and Some Energy Metabolism-Related Genes in the Longissimus Dorsi Muscle of Krskopolje Pigs: Effect of the Production System.

The Slovenian Krskopolje pig is the only preserved local autochthonous breed, appreciated mainly for its good meat quality and considered more appropriate for processing into dry-cured products. However, the biological characteristics of the skeletal myofibers of the Krskopolje breed, specifically the heavy myosin chain-based contractile and metabolic phenotypes that could affect meat quality, have not been established under different husbandry systems. The breed is generally maintained in either conventional indoor or organic systems. In the present study, the morphological, contractile, and metabolic properties of myofibers of the longissimus dorsi muscle were compared between animals reared in either an organic or a conventional indoor system. The myofibers were studied using immunohistochemical and succinate dehydrogenase (SDH) activity-based classification, histomorphometric assessment, and qPCR. Results revealed that the organic production system influenced the composition of the longissimus dorsi myofiber type, characterized by a smaller myofiber cross-sectional area, a shift toward oxidative (SDH-positive) myofiber types, increased relative expression of myosin heavy chain (MyHC) isoforms I, IIa, and IIx, and downregulation of MyHC IIb. On the contrary, no apparent effect was observed on the metabolic phenotype of the myofiber as assessed through relative mRNA expression of energy metabolism-related genes [peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor gamma (PPARγ), lipoprotein-lipase (LPL), carnitine palmitoyltransferase 1B (CPT1B), glycogen synthase 1 (GYS1), hexokinase 2 (HK2), and fatty acid synthase (FASN)]. Differences in MyHC expression were largely corroborated by the histochemical classification, indicating that the contractile protein content is directly regulated by the MyHC genes. A correlation between the muscle contractile and metabolic phenotypes was not established, except for that between the HK2 and MyHC I genes. In conclusion, the present study showed an evident effect of rearing on the longissimus dorsi myofiber contractile phenotype but not the metabolic phenotype. Moreover, obtained data suggest that rearing the Krskopolje pig breed in a conventional system would result in an increased fiber size and a greater proportion of type IIb myofibers, which are known to be negatively correlated with some meat quality traits.

Effect of immunocastration and housing conditions on pig carcass and meat quality traits.

The present study investigated the effects of immunocastration and housing conditions on carcass, meat, and fat quality traits. Immunocastrates (IC, n = 48), entire (EM, n = 48), and surgical castrates (SC, n = 48) male pigs were reared under three different housing conditions. The conditions were standard (n = 36), enriched (n = 36, twice as much space as standard and additional outdoor access), or standard with repeated social mixing (n = 72). Pigs of the IC group were vaccinated at the age of 12 and 22 wk. The animals were slaughtered in four batches, balanced for sex category and housing, at the age of 27 wk reaching 124.7 ± 1.0 kg. Immunocastration led to increased fat deposition (i.e., thicker subcutaneous fat at different anatomical locations, more leaf fat, fatter belly in IC than EM, P < 0.05) but did not affect muscularity traits. As a result, EM exhibited higher and SC lower (P < 0.05) carcass leanness than IC. Fatty acids composition of either subcutaneous or intramuscular fat (IMF) agreed with general adiposity, that is, IC were intermediate between EM and SC exhibiting the lowest and highest fat saturation (P < 0.05), respectively. Compared to SC, EM exhibited higher (P < 0.05) levels of muscle oxidation and collagen content than SC, with IC taking an intermediate position in the case of the level of peroxidation and collagen content, or closer to SC as regards to oxidation of muscle proteins (i.e., carbonyl groups). Meat quality (including marbling score, cooking loss, subjective color redness, and chroma) of IC was similar to EM, and both differed (P < 0.05) from SC. However, IC and SC had less (P < 0.05) tough meat than EM, consistent with protein oxidation. The effect of housing was less evident. Mixing of pigs resulted in lower (P < 0.05) carcass weight and fatness in all sex categories with lower (P < 0.05) oleic and higher (P < 0.05) arachidonic acid in IMF of EM.

Influence of Housing Conditions on Reliability of Immunocastration and Consequences for Growth Performance of Male Pigs.

Immunocastration is a sustainable alternative to piglet castration but faces limited market acceptance. The phenomenon of non-responders has not to date been examined in detail, but adverse and stressful housing conditions (e.g., mixing of groups) might impair the success of vaccinations. Therefore, we evaluated the influence of housing conditions on the immune response after two Improvac® vaccinations at an age of 12 and 22 weeks, respectively. Boars, immunocastrates and barrows (n = 48 each) were assigned to three different housing conditions (n = 36 enriched, n = 36 standard n = 72 repeated social mixing). Immune response was quantified by measuring GnRH-binding and its consequences for testosterone concentrations, development of the genital tract and boar taint. Growth performance was evaluated via average daily gain (ADG). GnRH-binding and testosterone levels revealed that immunocastration reliably suppressed testicular functions after the 2nd vaccination. Housing conditions did not modify testicular function but influenced ADG as animals under mixing grew slower than those under enriched conditions. Gonadal status had only a slight impact on ADG except in immunocastrates, which showed a temporarily higher ADG after the 2nd vaccination. The results show that immunocastration is a reliable procedure under different housing conditions and competitive in terms of growth performance.

Lactational exposure to dioxin-like polychlorinated biphenyl 169 and nondioxin-like polychlorinated biphenyl 155: Effects on rat femur growth, biomechanics and mineral composition.

Exposure to polychlorinated biphenyls (PCBs), which are persistent lipophilic environmental pollutants, has a variety of adverse effects on wildlife and human health, including bone mineralization, growth and mechanical strength. The present study evaluated the effects of lactational exposure to nondioxin-like PCB-155 and dioxin-like PCB-169, individually and in combination, on pubertal rat femur development and its biomechanics. After offspring delivery, Wistar rat mothers were divided into four groups, i.e., PCB-169, PCB-155, PCB-155+169 and control, and were administered PCBs intraperitoneally. Data on bone geometry, biomechanics and mineral composition were obtained by analysis of femurs from 42-day-old offspring by microCT scanning, three-point bending test and inductively coupled plasma mass spectrometry. Decreased somatic mass and femur size, i.e., mass, periosteal circumference and cross sectional area, were observed in the PCB-169 and PCB-155 groups. Additionally, lactational exposure to planar PCB-169 resulted in harder and more brittle bones containing higher amounts of minerals. Combined exposure to structurally and functionally different PCBs demonstrated only mild alterations in bone width and mineralization. To conclude, our results demonstrated that alterations, observed on postnatal day 42, were primarily induced by PCB-169, while toxicity from both of the individual congeners may have been reduced in the combined group.

Age-Dependent Expression of MyHC Isoforms and Lipid Metabolism-Related Genes in the Longissimus Dorsi Muscle of Wild and Domestic Pigs.

This study aimed to compare age-dependent changes in the relative expression of genes encoding myosin heavy chain (MyHC) isoforms and selected lipid metabolism-related genes in the longissimus dorsi muscle of wild pigs (WPs) and domestic pigs (DPs). Muscles sampled from postnatal day one as well as three-week-old and two-year-old animals were used in quantitative polymerase chain reaction (qPCR) assays, histological evaluations of succinate dehydrogenase (SDH) activity, and intra-myofiber lipid (IMFL) assessment. Expression of the MyHC isoforms displayed the most extensive age- and breed-dependent changes within the first three postnatal weeks. The MyHCembry level decreased significantly faster in the WPs than in the DPs. The relative MyHC-I and -IIa expression was significantly higher in the WPs, and MyHC-IIb was substantially higher in the DPs. The differences in MyHC expression corroborated the number of SDH-positive myofibers and IMFLs. Expression of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) genes displayed only age-related variations. In summary, the evidence is provided for accelerated postnatal myofiber transformation directed towards oxidative myofibers in WPs. The SDH activity/staining intensity largely reflected the expression of MyHCs, and not genes involved in lipid uptake and utilization.

Alterations in geometry, biomechanics, and mineral composition of juvenile rat femur induced by nonplanar PCB-155 and/or planar PCB-169.

Exposure to widespread lipophilic and bioaccumulative polychlorinated biphenyls (PCBs) induces diverse biochemical and toxicological responses in various organs, including the bone. The aim of this study was to evaluate the changes in growth rate, geometry, serum, and bone biochemical parameters and biomechanics of juvenile rat femur induced by lactational exposure to nonplanar PCB-155 and planar PCB-169 individually and in combination. Fifteen lactating Wistar rats were divided into four groups (PCB-169, PCB-155, PCB-155+169, and control), and PCBs were administered intraperitoneally at different time points after delivery. Femurs from 22-day-old offspring were analyzed by microCT, three-point bending test and inductively coupled plasma-mass spectrometry (ICP-MS) to obtain data on bone geometry, biomechanics and mineral composition. The serum levels of calcium, phosphate and alkaline phosphatase were also determined. Lactational exposure to planar PCB-169 resulted in shorter and thinner femurs, reduced endosteal and periosteal perimeters, smaller total cross-sectional and medullary areas, and lowered serum bone marker levels and calcium levels in the bone, while femur mechanical properties were not significantly altered. The changes observed in the combination exposure (PCB-155+169) group were similar to those observed in the PCB-169 group but were less pronounced. In summary, our results demonstrate that alterations in lactationally exposed offspring were primarily induced by planar PCB-169. The milder outcome in the combined group suggested that the PCB-169-mediated toxic effects on the bone might be reduced by a nonplanar PCB-155 congener. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1135-1146, 2017.

Effect of hydrolysable tannins on intestinal morphology, proliferation and apoptosis in entire male pigs.

This study aimed to evaluate the effect of hydrolysable tannin supplementation on morphology, cell proliferation and apoptosis in the intestine and liver of fattening boars. A total of 24 boars (Landrace × Large white) were assigned to four treatment groups: Control (fed commercial feed mixture) and three experimental groups fed the same diet supplemented with 1%, 2% and 3% of hydrolysable tannin-rich extract. Animals were housed individually with ad libitum access to feed and then slaughtered at 193 d of age and 122 ± 10 kg body weight. Diets supplemented with hydrolysable tannin affected the morphometric traits of the duodenum mucosa as reflected in increased villus height, villus perimeter and mucosal thickness. No effect was observed on other parts of the small intestine. In the large intestine, tannin supplementation reduced mitosis (in the caecum and descending colon) and apoptosis (in the caecum, ascending and descending colon). No detrimental effect of tannin supplementation on liver tissue was observed. The present findings suggest that supplementing boars with hydrolysable tannins at concentrations tested in this experiment has no unfavourable effects on intestinal morphology. On the contrary, it may alter cell debris production in the large intestine and thus reduce intestinal skatole production.

Elevated fat skatole levels in immunocastrated, surgically castrated and entire male pigs with acute dysentery.

Boar taint is due to androstenone and skatole (3-methyl-indole) accumulation in fat tissues. During a study to investigate the effect of immunocastration on fattening pigs, an outbreak of acute dysentery occurred caused by Lawsonia intracellularis and Brachyspira hyodysenteriae and resulted in cachexia and high mortality. Low androstenone levels in the immunocastrates (0.25 ± 0.04 µg/g liquid fat) suggested that the immunocastration had been effective, but unusually high skatole concentrations in fat tissues were found not only in entire males, but also in surgical castrates and immunocastrates (0.22 ± 0.15, 0.14 ± 0.08 and 0.18 ± 0.14 µg/g liquid fat, respectively). The findings suggest that boar taint can arise in cases of intestinal infections, even in castrated pigs.

Obesity resistant mechanisms in the Lean polygenic mouse model as indicated by liver transcriptome and expression of selected genes in skeletal muscle.

BACKGROUND: Divergently selected Lean and Fat mouse lines represent unique models for a polygenic form of resistance and susceptibility to obesity development. Previous research on these lines focused mainly on obesity-susceptible factors in the Fat line. This study aimed to examine the molecular basis of obesity-resistant mechanisms in the Lean line by analyzing various fat depots and organs, the liver transcriptome of selected metabolic pathways, plasma and lipid homeostasis and expression of selected skeletal muscle genes. RESULTS: Expression profiling using our custom Steroltalk v2 microarray demonstrated that Lean mice exhibit a higher hepatic expression of cholesterol biosynthesis genes compared to the Fat line, although this was not reflected in elevation of total plasma or liver cholesterol. However, FPLC analysis showed that protective HDL cholesterol was elevated in Lean mice. A significant difference between the strains was also found in bile acid metabolism. Lean mice had a higher expression of Cyp8b1, a regulatory enzyme of bile acid synthesis, and the Abcb11 bile acid transporter gene responsible for export of acids to the bile. Additionally, a higher content of blood circulating bile acids was observed in Lean mice. Elevated HDL and upregulation of some bile acids synthesis and transport genes suggests enhanced reverse cholesterol transport in the Lean line--the flux of cholesterol out of the body is higher which is compensated by upregulation of endogenous cholesterol biosynthesis. Increased skeletal muscle Il6 and Dio2 mRNA levels as well as increased activity of muscle succinic acid dehydrogenase (SDH) in the Lean mice demonstrates for the first time that changes in muscle energy metabolism play important role in the Lean line phenotype determination and corroborate our previous findings of increased physical activity and thermogenesis in this line. Finally, differential expression of Abcb11 and Dio2 identifies novel strong positional candidate genes as they map within the quantitative trait loci (QTL) regions detected previously in crosses between the Lean and Fat mice. CONCLUSION: We identified novel candidate molecular targets and metabolic changes which can at least in part explain resistance to obesity development in the Lean line. The major difference between the Lean and Fat mice was in increased liver cholesterol biosynthesis gene mRNA expression, bile acid metabolism and changes in selected muscle genes' expression in the Lean line. The liver Abcb11 and muscle Dio2 were identified as novel positional candidate genes to explain part of the phenotypic difference between the Lean and Fat lines.

Enzyme- and immunohistochemical aspects of skeletal muscle fibers in brown bear (Ursus arctos).

To further elucidate the pattern of MHC isoform expression in skeletal muscles of large mammals, in this study the skeletal muscles of brown bear, one of the largest mammalian predators with an extraordinary locomotor capacity, were analyzed. Fiber types in longissimus dorsi, triceps brachii caput longum, and rectus femoris muscles were determined according to the myofibrillar ATPase (mATPase) histochemistry and MHC isoform expression, revealed by a set of antibodies specific to MHC isoforms. The oxidative (SDH) and glycolytic enzyme (alpha-GPDH) capacity of fibers was demonstrated as well. By mATPase histochemistry five fiber types, i.e., I, IIC, IIA, IIAX, IIX were distinguished. Analyzing the MHC isoform expression, we assume that MHC-I, -IIa, and -IIx are expressed in the muscles of adolescent bears. MHC-I isoform was expressed in Type-I fibers and coexpressed with presumably -IIa isoform, in Type-IIC fibers. Surprisingly, two antibodies specific to rat MHC-IIa stained those fast fibers, that were histochemically and immunohistochemically classified as Type IIX. This assumption was additionally confirmed by complete absence of fiber staining with antibody specific to rat MHC-IIb and all fast fiber staining with antibody that according to our experience recognizes MHC-IIa and -IIx of rat. Furthermore, quite high-oxidative capacity of all fast fiber types and their weak glycolytic capacity also imply for MHC-IIa and -IIx isoform expression in fast fibers of bear. However, in adult, full-grown animal, only MHC-I and MHC-IIa isoforms were expressed. The expression of only two fast isoforms in bear, like in many other large mammals (humans, cat, dog, goat, cattle, and horse) obviously meets the weight-bearing and locomotor demands of these mammals.

Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth.

To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (alpha-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4-6 weeks of age, which coincides with the increased physical activity of puppies.