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ANIMAL NUTRITION |
Division of Nutritional Sciences, School of Biosciences, Sutton Bonington Campus, The University of Nottingham, Leicestershire, LE12 5RD, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Key Words: Maternal Nutrition • Muscle Fiber Formation • Sheep
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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). In some species, the total number of muscle fibers for life is essentially set during fetal life (Ashmere et al., 1972), and, therefore, to achieve maximal skeletal muscle fiber number, the animal requires optimal conditions for muscle development during fetal life, which includes an adequate supply of nutrients. The fetus obviously depends on maternal nutrition during gestation, a decrease of which may result in decreased myoblast proliferation coupled with an earlier onset of differentiation to fibers (Brameld et al., 2000). The aim of this study was to test the hypothesis that maternal undernutrition before muscle fiber formation will decrease the number of muscle fibers in the offspring. Previous work (Fahey et al., 2005) indicated that, in sheep, muscle cell proliferation occurs before d 85, and differentiation commences around d 85. These findings were determined by observing the change in expression of IGF-I and -II, growth hormone receptor, myostatin, and myogenin, as these are factors known to influence myoblast proliferation and differentiation. This knowledge of muscle development led to the design of the present study. One group of the ewes was restricted before muscle fiber formation (d 30 through 70 of gestation), one group was restricted during fiber formation (d 55 through 95 of gestation), and one group was restricted after fiber formation (d 85 through 115 of gestation). By restricting the ewes at different periods in gestation, a clear indication would be obtained as to not only if, but when during gestation muscle development can be altered by maternal nutrient restriction. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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). The quantity of diet given to the ewes throughout this trial was always a proportion of their daily requirement to support the ewe and allow for conceptus growth; subsequently, throughout the text, this quantity will be referred to as their daily requirement. The diet was fed in two equal rations at 0800 and 1600 and supplied 8.6 MJ/d at the start of the trial until d 90 of gestation. Ewes were given water ad libitum throughout the trial. Any orts were weighed and recorded. At d 30, the ewes were scanned using an ultrasound scanner (OVISCAN 4 Sector Scanner; BCF Technology Ltd., West Lothian, Scotland, UK). Those carrying twins were then allocated randomly into one of four groups. Group d 30–70 (n = 8) ewes were fed 100% of their daily requirement until d 30, when their diet was restricted to 50% for a period of 40 d (d 70); they were then returned to 100% of their daily requirement until term (d 150). Group d 55–95 (n = 8) ewes were fed 100% of their daily requirement until d 55, when their diet was restricted to 50% for a period of 40 d (d 95); they were then returned to 100% of their daily requirement until term (d 150). Group d 85–115 (n = 8) ewes were fed 100% of their daily requirement until d 85, when their diet was restricted to 50% for a period of 30 d (d 115); this period was shorter than for the other two treatment groups to prevent the onset of pregnancy toxemia (twin lamb disease). The control group (n = 8) was fed 100% of their daily requirement throughout gestation. The quantity of vitamins and minerals was kept constant throughout, even during the periods of restriction. The ewes were weighed twice weekly, and the diet was adjusted according to live BW changes. At d 90 and 100, the intakes were increased by the addition of barley (200 and 400 g/d at d 90 and 100, respectively) so that the ewes received 12.27 and 14.54 MJ/d respectively, for a 75-kg ewe (AFRC, 1993) when fed 100% of their daily requirement. The restricted ewes were fed 50% of the increased energy values. Ewes have a greater energy requirement in the last trimester of gestation because of the increasing demands of the developing fetus; therefore, from d 100 until term, 100% of their daily requirement was taken to be 14.54 MJ/d. After parturition, the lactating ewes were fed a commercial diet (Frank Wright Ltd.). Quantities given were calculated on an individual ewe basis according to BW.
Sample Collection
Birth weight, sex, and order of birth of each lamb were recorded, and where possible, the placental weight and number of placentomes were also recorded. Each ewe was left to deliver the placenta naturally; care was taken to ensure that any placental weights taken were of the total placenta with any fluid being removed before weighing. The number of placentomes also was counted and recorded.
On d 14 (after parturition), the lambs were slaughtered by an overdose of anesthetic sodium pentobarbitone (Vetoquinol UK Ltd, Bicester, UK: 130 ng/kg of BW) using the recommended dose. Samples of the LM, semitendinosus (ST), and vastus lateralis (VL) muscles were dissected. A sample of each muscle was snap frozen in dry ice-chilled isopentane. These samples would be used for muscle histology analysis, whereas samples needed for protein work were snap frozen in liquid N2 alone. All samples were stored at –70° C, and the whole weight of each muscle was recorded.
Myosin ATPase Staining Technique
The different fiber types within a muscle were determined using myosin ATPase staining. Serial tissue sections (20-µm thick) obtained from cryostat sectioning of samples that had been frozen in dry ice-cooled isopentane were stained for myosin ATPase after pre-incubation at pH 4.6 and 10.4 at room temperature. The slow type I fibers are stable in acid and therefore stain dark following incubation in an acidic buffer, whereas the fast type II fibers are labile in an acidic solution and stain light. Conversely, the slow type I fibers are unstable in an alkaline condition and will stain light, whereas the fast type II fibers are stable in an alkaline condition and will stain dark after preincubation in an alkaline buffer (Brooke and Kaiser, 1970). The acid preincubation (pH 4.6) consisted of 8 min in 26.4 mM sodium acetate buffer. Another section of the same muscle that had been taken from the same serial sectioning was preincubated for 8 min in 36 mM CaCl2 buffered with 100 mM Tris·HCl, pH 10.4 (alkaline buffer). Sections were then washed 3 times for 1 min each time with 18 mM CaCl2, 100 mM Tris·HCl (pH 7.8). Sections were then incubated for 45 min at 37° C in 18 mM CaCl2, 100 mM Tris HCl, and 4.5 mM ATP (pH 9.4). After the incubation, sections were washed twice for 30 s each time in 1% CaCl2 solution followed by 3 min of incubation in 2% CoCl2 solution at room temperature. Sections were then washed in distilled water before being stained with 1% ammonium sulfide (wt/vol; Sigma-Ald-rich, Dorset, UK) solution for 3 min. The stained sections were finally washed with distilled water, and a cover slip was mounted onto each slide before viewing under a light microscope. To facilitate the counting of the different fiber types, the sections were viewed on a light microscope (200x; Nikon, Japan). Eight areas were selected randomly from the serial sections of each sample. Within this area, the numbers of slow fibers and fast fibers were counted, and the diameter of individual muscle fibers was measured for each muscle fiber type. The muscle fiber diameter was measured from approximately 20 fibers of each fiber type from each area counted.
Extraction of Muscle Protein
Muscle samples that had been snap frozen in liquid N2 were finely ground using a pestle and mortar that had been cooled using liquid N2. One gram of the fine muscle powder was then homogenized in 10 mL of Buffer A (20 mM Tris, 5 mM EDTA, adjusted to pH 7.5 with 1 M HCl), which contained 200 µg AEBSF (2-(4-aminoethyl)-benzenesulphonyl fluoride)/mL, leupeptin (1 µg/mL), and pepstatin (1 µg/mL) to protect against proteolytic digestion of the protein samples. The muscle homogenates were diluted twofold in 2x SDS mix (20% glycerol, 125 mM Tris [pH 6.8], 4% SDS, 100 mM ß-mercaptoethanol, 0.2% bromophenol blue). The samples were stored at –20° C.
Western Blot Analysis of Myosin Heavy-Chain Slow and Fast
The expression of the proteins myosin heavy-chain slow (MHC-slow) and fast (MHC-fast) at 205 to 250 kDa and actin at 42 to 45 kDa (as an internal standard) were measured in whole muscle homogenates using immunoblotting. The samples were separated on 8% SDS-PAGE (Laemmli, 1970) and electroblotted onto a nitrocellulose membrane that was then cut to enable both myosin and actin to be probed independently at the same time. The part of the membrane containing myosin was either probed with anti-human MHC-slow (1:1,000 dilution) or anti-human MHC-fast (1:1,000 dilution) monoclonal antibodies (Novacastra Laboratories, Newcastle upon Tyne, UK) at room temperature for 45 min. The part of the membrane containing actin was subjected to rabbit anti-actin antibody (Sigma-Ald-rich; 1:500 dilution) probing at room temperature for 45 min. Membranes incubated with the anti-MHC primary antibodies were subsequently incubated with anti-mouse IgG conjugated with alkaline phosphatase (1:5,000; Amersham Biotech, Buckinghamshire, UK), whereas membranes probed with the primary anti-actin antibody were incubated with anti-rabbit IgG conjugated with alkaline phosphatase (1:5,000; Sigma-Ald-rich), each at room temperature for 30 min. The bands on membranes were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham Biotech). Because of the number of animals in the trial, not all samples could be run on the same gel; therefore, a number of separate blots were involved in each analysis. As a result, the same sample was distributed across each gel to normalize variation between gels, which might arise because of changes in the length of exposure time and the blotting/immunoprobing efficiency.
Data Analyses
The number of slow and fast fibers in LM, ST, and VL were measured by counting the number of slow and fast fibers in eight randomly selected areas from the serial sections of each muscle sample. The muscle fiber diameter was measured from approximately 20 fibers of each muscle type from each area counted. The intensity of the MHC-slow, MHC-fast, and actin bands were quantified using Multi Analyst image software (Bio Rad, Hercules, CA). The effect of maternal undernutrition during gestation on the number and diameter of fast and slow fibers and the levels of MHC-slow and MHC-fast were analyzed using a general linear regression and one-way ANOVA. A general linear model was carried out on the data, which showed that any variation in the results caused by the lambs coming from different ewes (within a treatment group) was not significant (P = 0.73). Therefore, in this study, means were based on the number of lambs, not the number of ewes, because each lamb was taken to be independent from one another. The statistical software used was Genstat (Version 6; Lawes Agricultural Trust, Hertfordshire, UK). Data were considered significant when P < 0.05.
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Results |
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). A similar trend was noted when observing the weight of the individual muscles (LM, ST, and VL; Table 1). The weight of each muscle was decreased in lambs born from ewes restricted between d 85 through 115 compared with the other groups. In Group d 85–115, the decrease in weight was significant in the VL (P < 0.01) and LM (P < 0.05), whereas the decrease observed in ST muscle tended (P = 0.08) to be significant (Table 1). There was a difference observed between the birth weights depending on whether the lamb was male or female (P < 0.05). Female lambs weighed slightly less at birth (data not shown). There was no significant difference between treatment groups in the growth rate of the lambs over the 2 wk from birth to slaughter; however, a difference (P < 0.05) was observed in the growth rate between the two sexes. Female lambs grow at a slower rate (data not shown). Because a difference was observed between the sexes for both birth weight and growth rate, a general linear model was performed on the data to compare whether the maternal restriction affected one sex in particular. The sex of the twin’s sibling in utero also was taken into consideration; however, no difference between the sexes was observed for muscle fiber number (P = 0.48) or diameter (P = 0.72). Hence, the data are not shown by sex.
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). A significant difference was observed, however, between the treatment groups with regard to the number of placentomes per placenta (Table 1). Ewes in Group d 30–70 were observed to have more (P < 0.001) placentomes than those in the other treatment groups.
Myosin Heavy-Chain Expression
Results of Western-blot analysis indicated a significantly greater MHC-slow content (P < 0.05) in the LM muscle samples taken from the lambs born to the ewes in Group d 30–70 compared with those from the other treatment groups. This was not as clear for the VL because, although an increase (P < 0.05) in MHC-slow expression was observed in the lambs born from the ewes in Group d 30–70 compared with the control ewes and ewes in Group d 85–115, this increase was not significant when compared with lambs born from ewes in Group d 55–95. The same trend that was observed for the LM also was noted for ST, but the results were not significant (Table 2).
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) in both the LM and VL from lambs born to the ewes in Group d 30–70 compared with the other treatment groups. Again, the same trend was observed for the ST; however, the results were not significant (P = 0.29; Table 2).
Slow Fiber Number
Eight areas were selected randomly from the serial sections of each muscle sample. Within this area, the number of slow fibers was counted. The LM (P < 0.001) and VL (P < 0.001) were both observed to have significantly more slow fibers in the samples taken from the lambs born from the ewes in Group d 30–70 compared with the other treatment groups. Semitendinosus also tended to have more slow fibers (P = 0.06) from the same group of lambs (Table 3).
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).
Muscle Fiber Diameter
Within each area counted, the diameters of approximately 20 fibers of each fiber type were measured. The diameter of the slow muscle fibers in LM, ST, and VL were not affected by maternal nutrition, as no significant difference in slow fiber diameter was observed between the lambs born from the different treatment groups (Table 3). Nonetheless, a significant difference was observed in the fast fiber diameter. Lambs born from the ewes in Group d 30–70 had fast fibers with a greater diameter in the LM (P < 0.001) than those born from the ewes in the other treatment groups. A significant increase in fast diameter also was observed in the VL (P < 0.05) in the lambs born from ewes in Group d 30–70 compared with both the control group and Group d 85–115. Although fast fiber diameters were numerically greater in the lambs born from the ewes in Group d 30–70 compared with the lambs born from ewes in Group d 55–95, the increase was not significant (P = 0.25). No significant change was observed in ST fast fiber diameter (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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). Therefore, it would seem that increases in muscle fiber size are more important than fiber number in contributing to postnatal increases in muscle mass; however, this is not the case. The number of muscle fibers within a muscle has been found to be of high importance for the growth potential of skeletal muscle, for endurance fitness, and for adaptability to environmental stress. In farm animals, muscle fiber number also is important for meat content and quality (Rehfeldt et al., 1999). It is thought that, although the fetal genome may determine the growth potential of an offspring in utero, the growth that is actually achieved is primarily determined by environmental effects, such as the availability of nutrients to the fetus (Godfrey and Barker, 2001). Consequently, the aim of this study was to test the hypothesis that decreased maternal nutrition before muscle fiber formation would reduce the number of muscle fibers in the offspring. Results of the present study indicate that by restricting the maternal diet to 50% of the daily requirement between d 30 and 70 of gestation, muscle fiber development was altered and resulted in a decrease in the number of fast muscle fibers. Changes noted in the ST muscle were generally not significant. A study by Greenwood et al. (2000) evaluating the effect of birth weight and postnatal growth in neonatal sheep also concluded that not all muscles are affected equally by variation in prenatal and post-natal nutrition.
The results of Western-blot analysis indicated a significantly greater MHC-slow content (P < 0.05) in the LM samples taken from the lambs born to the ewes in Group d 30–70 compared with those from the other treatment groups. This was not as clear for the VL because, although a significant increase in MHC-slow expression was observed in lambs born from ewes in Group d 30–70 (P < 0.001) compared with lambs born from ewes in the control group and Group d 85–115), this increase was not different from the MHC-slow content measured in lambs in Group d 55–95. The same was noted when observing the fast fiber diameter of the VL muscle. One possible reason for this discrepancy in the VL muscle might be due to a difference in the period during gestation at which this particular muscle develops. Perhaps the VL muscle develops at a slightly later time than the LM. The nutrient restriction that occurred between d 55 and 95 might, therefore, have been during a time that is still before the fiber formation of this muscle and is thus a period that is susceptible to manipulation. In contrast, the fast fiber diameters of the VL muscle were larger than those of the LM in lambs from the control group (Table 3), which is not consistent with the previous statement. This finding may indicate that the VL is actually maturing earlier than the LM. The results from this study show that it would be wrong to conclude that all muscles from one species develop at the same time. In the present study, changes in the ST muscle were generally not altered. Perhaps the maternal restriction did not occur at a critical time for this muscle, or its muscle fiber formation transpired at a slightly different time than other muscles observed in this study.
McCoard et al. (2000) showed a decrease in the skeletal muscle mass of the twin fetuses compared with single fetuses at 140 d of gestation; however, the myofiber number did not differ. The similar myofiber number but smaller muscle mass between the twins and singletons suggests that competition between littermates does not affect the structural and functional integrity of the muscles, but only the muscle hypertrophy. The nutrient restriction to the twin fetuses should have only been moderate; therefore, this study highlights the ease with which nutrition during gestation can affect myofiber hypertrophy and the importance of adequate fetal nutrition for skeletal muscle growth in sheep. There was a significant decrease in the birth weight of lambs born from the ewes in Group d 85–115 compared with lambs born from ewes in the other treatment groups. A similar trend was noted when observing the weight of the individual muscles (LM, ST, and VL). With each muscle, the weight decreased in the lambs born from the ewes in Group d 85–115 compared with the other groups. This decrease in weight was significant in the VL and LM, and the decrease observed in ST muscle tended (P = 0.08) to be significant. The birth weights and the weights of the individual muscles suggest that nutrient restriction in the last trimester seems to have an effect on fetal growth (not development). It seems that early restriction affects the development of ovine fetal muscle, and late nutrient restriction affects the hypertrophy of the already developed muscle. When the weight of the muscles was analyzed relative to the BW of the lamb at slaughter, any significant changes for the LM and ST were lost (Table 1), suggesting that the nutrient restriction later in gestation had not specifically affected the muscles of the animal, although nutrient restriction caused a general loss in full BW. However, when the weight of the VL was analyzed relative to the BW of the lamb, the significant changes were still observed (Table 1), which perhaps suggests that nutrient restriction in late gestation specifically affected this particular muscle. Total muscle mass of a lamb at this age (2 wk) would represent a large percentage of the total body mass.
From the results of the present study, it is evident that the timing of nutrient restriction is critical in determining fetal response. A nutrient restriction during early gestation seemed to affect hyperplasia of the muscle cells, whereas a restriction later in gestation resulted in lighter muscles, indicating that hypertrophy of the muscle was affected. Will any differences carry on into adult life? Is it likely that the carcass composition of the lambs that were born from ewes that were restricted during early gestation will differ from those that were born from ewes restricted in late gestation? Both of these questions can only be answered by further research.
The ewes in this study that were restricted from d 30 until d 70 were observed to have significantly more placentomes than those in the other treatment groups. This result is consistent with previous work (Clarke et al., 1998), which also has shown that a restriction in maternal nutrition in early to mid gestation led to an increase in the number of placentomes per placenta. In contrast, Osgerby et al. (2002) found a difference in the placental weights, with undernourished pregnant ewes having lighter placentas than well-fed pregnant ewes. The maternal undernutrition in their study was for most of gestation (d 26 through 135), and this could be the reason for the differences between their study and the present experiment. All of the restrictions in the present study were for 
40 d, after which the diet was returned to 100% of daily requirement. Perhaps this period of adequate nutrition provided the placenta with enough nutrients to achieve a normal weight by birth. The development of the placenta occurs during early to mid gestation, and therefore the change in the number of placentomes because of maternal nutrient restriction would be determined early in gestation and would most likely not be altered by adequate nutrition later in gestation.
The lambs born from the ewes restricted between d 30 through 70 of gestation were observed to have significantly fewer fast fibers and significantly more slow fibers in both the LM and VL compared with the other groups. Previous studies (Wilson et al., 1988; Dwyer and Stickland, 1992) have shown that primary fibers are resistant to environmental factors such as nutrition and that secondary fibers are preferentially affected. As already stated, primary fibers tend to form slow fibers; therefore, it could be suggested that there are not more slow fibers in the ovine muscle samples, although there are proportionally more because of the decrease in fast fibers. Undernutrition of pregnant sows leads to offspring with fewer secondary muscle fibers but no alteration of primary fibers (Dwyer and Stickland, 1991). Low birth weight caused by multiple offspring in the pig also was associated with a lower number of secondary fibers (Handel and Stickland, 1987). On the contrary, overnutrition of the sow during early gestation has been observed to increase the total number of muscle fibers in the offspring (Dwyer et al., 1994). The primary fiber number was unaltered, and the increase was due to increases in the secondary fiber number. It is evident from the literature that any changes in the muscle fiber formation will most likely be due to changes in the secondary fiber number. Therefore, we decided not to address primary and secondary fiber formation separately in this study but to focus on fiber formation as a whole. Neither the immunoblotting nor the histochemical methods used in the present experiment enabled subgroups of the type II fibers (type IIA, type IIB, and type IIC) to be distinguished. The aim of this study was not to observe a particular fiber type but to observe the effect of maternal nutrition on muscle fiber number of the offspring. Results of this study indicate that by restricting the maternal diet to 50% of the ewe’s daily requirement between d 30 and 70 gestation, muscle fiber development was altered, resulting in a decrease in the number of fast muscle fibers.
Conclusions
The results have shown that decreased maternal diet before fiber formation will alter the muscle fiber development in the fetus. It is clear that this change in muscle development is not only caused by the nutrient restriction but that the timing of the insult also is important in determining the fetal response. In farm animals, muscle fiber number and fiber type are seen to influence meat content and quality. Further studies should help determine whether the change in muscle fiber development caused by maternal undernutrition affects the meat or carcass quality of the subsequent offspring.
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Footnotes |
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2 A. Fahey was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship.
3 Correspondence—phone: 44 115 951 6137; fax: 44 115 951 6120; e-mail: Peter.Buttery{at}nottingham.ac.uk.
Received for publication December 1, 2004. Accepted for publication July 12, 2005.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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