Differences in muscle characteristics of piglets related to the sow parity

The total number of born piglets increases with parities, but the higher prolificacy results in lower individual average birth weights and more heterogeneous litters. Variations in piglets’ birthweights are related to their number of muscular fibers resulting in variable performances after birth (Rehfeldt and Kuhn 2006).

The skeletal muscle in mammals begins to be developed in the embryonic stage, and continues during the fetal and the post-natal phases, corresponding to primary, secondary and post-natal myogenesis, respectively. Ashmore et al. (1973) found that the primary muscle fibers are already present in the pig fetus at 35 days of gestation and Wigmore and Stickland (1983) had shown that the hyperplasia of the muscular fibers is completed at about 85 to 90 days of gestation.

There are three different types of muscle fibers, called primary and secondary fibers and satellite cells. Dwyer et al. (1994) had shown that the primary muscle fibers represent only a small proportion in the total number of muscle fibers, ranging about 5%. However, these fibers act as a base for the development of the secondary muscle fibers. The surface of primary fibers is proportional to the number of secondary muscle fibers which represents about 95% of the total number of fibers in the muscle (Kelly and Zacks 1969).

According to Wigmore and Stickland (1983) and Foxcroft et al. (2006), primary muscle fibers are resistant to environmental changes whereas secondary fibers are susceptible to a large range of pre-natal events related mainly to nutritional factors and intra-uterine environment during pregnancy.

Given that sow’s parity influences litter size and piglets’ birthweight, this study was designed to test the hypothesis that differences in muscle characteristics of piglets are affected by parity and also that secondary muscle fiber number could affect piglets’ birthweight.

Material and Methods

Animals were used and cared for in accordance with the Committee of Ethics in Animal Use (CEUA) no. 26368.2012.23.

Eighty-one Topigs_Danbred sows from the 1st to the 7th parity and their litters were used to identify the differences in the semitendinosus muscle characteristics of piglets according to parity of sows. The sows were identified by ear tags and were given similar diets and husbandry conditions. Estrus detection was done by passing a young boar (8 to 12 mo of age) twice daily (between 0800 and 0900 and from 1600 to 1700). When estrus was detected, sows were inseminated at 24, 36 and 48 h after estrus and gilts were inseminated at 12, 24 and 36 h after estrus. For practical reasons, this timing was not strictly followed, but was quite close. Insemination was done within a week with 85 mL of semen (3_109 live sperm cells) from different boars distributed homogeneously over all sows in order to avoid any boar effect.

The sows were allocated into individual cages during gestation and into maternity pens during lactation. All of them had similar backfat thickness (determined using P2 ultrasonic measurement) and feed intake during gestation was restricted at 2.5 kg_1 animal_1 d_1 until 84 d of gestation and 3.5 kg animal_1 d_1 between 85 and 114 d of gestation. At 111 d of pregnancy, the sows were transferred to the maternity pen. They received no feed on the day of delivery. The diet is described on Table 1.

Piglets’ birth weight (within 5 min after birth), gender, total number of piglets born, and the numbers of piglets born alive were recorded. The litter’s average weight was calculated, and the piglets with weights equal to or near to the litter average weight were sacrificed (three piglets per sow, totaling 233 piglets) for the collection of the semitendinosus muscles. The muscles were weighed and stored for 24 h in Bouin’s solution for fixation and later preserved in 70% alcohol.

The muscle samples were submitted to dehydration in solutions of increasing concentrations of alcohol, diaphanized in xylol using an automatic tissue processor Leica TP1020, embedded in paraffin, and then mounted on histological slides. Sections 5 mm thick were cut from the middle portion of the muscle using a rotary microtome LAB-MR500 and stained with hematoxylin and eosin for histological evaluation. Motic Images Plus 2.0 software was used to obtain the images and to measure the area of the semitendinosus muscle, and the number of secondary muscle fibers was estimated by counting eight randomly selected fields (36.7 mm2 per field) from each slide using an Olympus BX 50 microscope. Knowing the total area of the muscle, the area of the field, and the average number of fibers per fields, it was possible to estimate the total number of secondary muscle fibers in the whole muscle by simple multiplying these variables.

Table 1. Composition of experimental diets used for the gestation (0 d gestation until parturition) period

Differences in muscle characteristics of piglets related to the sow parity - Image 1

^zPrevitamin and mineral mix (nutrient per kilogram product): vitamin A, 1250.00 IU; vitamin D₃, 250 000 IU; vitamin E, 8.750 mg; vitamin K₃, 150 mg; vitamin B₁, 125 mg; vitamin B₂, 1.125 mg; vitamin B₆, 150 mg; vitamin B₁₂, 4500 mcg; niacin, 3750 mg; calcium pantothenate, 3.250 mg; folic acid, 400 mg; biotin, 50 mg; choline chloride, 75 g; Fe, 12.25 g; Cu, 5.250 mg; Mn, 8.750 mg; Zn, 26.25 g; iodine, 350 mg; selenium, 5 mg kg^_1.

Data were analyzed using SAS software (Littell et al. 1996) as a completely randomized design, with unequal frequencies. Homogeneity of the data was tested using Bartlett’s Test for homogeneity of variance. To evaluate the effect of parity on each characteristic, regression trials were done considering the sow as an experimental unit (the average of the three piglets euthanized per sow) and the correlation between characteristics was tested by Pearson Correlation. For the evaluation of gender and weight class on muscle fiber number, a 3_2 factorial arrangement (weight class and gender) was used. The model was:

where Yij is the dependent variable, m is a constant; Ti is the weight classs effect, Sj is the gender effect, TSij is the weight class and gender interaction, and eij is the residual error. When treatment effects were significant a Tukey test at 0.05% was performed, considering each piglet as an experimental unit and gender and weight class as treatments.

Results and discussion

Several reports have studied the impact of nutrition during gestation on fetal and post-natal development (Wigmore and Stickland 1983; Dwyer et al. 1994; Musser et al. 2004; Nissen et al. 2003; Gatford et al. 2003; Bee 2004; Ferguson et al. 2006; Karunaratne et al. 2007; Cerisuelo et al. 2009; Rehfeldt et al. 2012) as well as line, gender, litter size and additive genetic variation (Miller et al. 1975) on porcine muscle development. The present results present a new perspective in this regard _ the effect of parity.

No significant effect of gender was found for body weight of piglets, number of muscle fibers, or semitendinosus muscle weight and area at birth. The effect of gender on muscle area is controversial. Some authors have shown larger muscle areas in gilts than in barrows (Solomon et al. 1990; Larzul et al. 1997), whereas others found no differences (Sosnicki 1987; Ender 1994). According to Bee et al. (2004), these results illustrate the difficulty in sampling procedures and analyses techniques for myofiber counting due to the high subjectivity.

A quadratic effect of parity was found on the semitendinosus muscle weight of the piglets at birth (PB0.05; R2_0.44), the semitendinosus muscle area (PB0.05; R2_0.64), and the number of secondary muscle fibers (PB0.05; R2_0.54) (Fig. 1).

Due to the larger number of muscle fibers to be formed, the secondary myogenesis can vary substantially (Zhu et al. 2008) depending on the sow’s age and parity, the genotype, the placental and uterine size and functional capacity, the uteroplacental transfer of nutrients and oxygen, and the metabolic and hormonal status of the embryo (Ashworth et al. 2001; Reynolds et al. 2005). Young sows (parities 1 and 2) are still growing during their gestation and, therefore, they have proportionally higher energy and protein requirements than adult sows (Eissen et al. 2000). In this regard, parity can affect the partition of nutrients between the maternal and the embryonic tissue, and prenatal undernutrition is related to lower muscle fiber numbers, myonuclear numbers, or muscle DNA content (Wigmore and Stickland 1983; Handel and Stickland 1987; Dwyer and Stickland 1991; Rehfeldt et al. 2001).

Differences in muscle characteristics of piglets related to the sow parity - Image 3

Fig. 1. Influence of parity on (a) the semitendinosus muscle weight (y_2.57481_0.393311 x _ 0.06207 x², R²_0.44); (b) the semitendinosus muscle area (y_55.6516_6.01861 x _ 0.928982 x², R²_0.64), and (c) the number of secondary muscle fibers (y_349816_28102.9 x _ 3747.45 x², R²_0.54).

Also, considering that the skeletal muscle is not a priority during the early development of embryos (Zhu et al. 2008), it is reasonable to suggest that embryo requirements for muscle development are not met during late gestation, as suggested by Nissen et al. (2004), in young sows, resulting in an impaired development of the muscular tissue when compared with 3rd and 4th parity sows.

Intrauterine crowding could be the main cause of this impairment in high parity sows (parities 5, 6, 7). Town et al. (2004) have suggested that intrauterine crowding on the 30th day of gestation can affect muscle fiber differentiation through a down expression of some myogenesis-regulating factors, and Foxcroft and Town (2004) hypothesized that intrauterine crowding could affect the development of fetal organs and muscle fiber and type, as intrauterine growth retardation does.

There were positive correlations for the weight of the semitendinosus muscle and the number of muscle fibers (PB0.05; 0.39), for the area of the muscle and the number of muscle fibers (PB0.001; 0.63) and for muscle area and semitendinosus muscle weight (PB 0.001; 0.64).

Together, these results suggest that 3rd and 4th parity sows are not affected, or are less affected, by factors impairing fetal growth, resulting in better fetal muscular characteristics, in particular muscle weight and muscle area, which improves fetal development by a higher muscle growth rate due to better protein turnover, as shown by Nissen et al. (2004).

Parity had a quadratic effect (y_11.2174_2.3573 x _ 0.20489 x2, R2_0.66) on the total number of piglets born, on average birthweight (PB0.05; R2_0.54), and a tendency to a linear effect (y_13.02_0.268931 x, R_0.07) on the number of piglets born alive (Table 2). It is known that parity influences productivity, mainly because of the number of piglets born alive and the average litter weight (Holanda et al. 2005; Cavalcante- Neto et al. 2011). Our results partially agree with those of Mahan and Peters (2004), who found that the total pigs born tended to increase as parity increased, but there was parity effect on the number of live pigs, and litter and 0-d-old pig weights increased as parity increased.

Table 2. The influence of parity on the total number of piglets born per litter, number of piglets born alive, average birth weight, muscle fiber numbers, semitendinosus muscle weight and areaz

Differences in muscle characteristics of piglets related to the sow parity - Image 4

^zWithin a row, there is a quadratic effect as parity increases (PB0.05). ^yTBL, total born per litter; TBA, born alive; TLB, total litter birth weight; ABW, average birth weight; MFN, muscle fibers number; MW, muscle weight; MA, muscle area.

The highest litter birth weight was achieved by 4th parity sows, with 1.16% and 5.24% differences compared with 5th and 3rd parity sows, the second and third best results, respectively, being 4.03% the difference between them. The higher litter birth weight for 5th parity sows when compared with 3rd parity sows was due to the total number of piglets born, which was 9.15% higher. In addition, the highest average birth weight was found in 3rd parity sows (5.56% higher tan 5th parity sows), followed by 4th and 2nd parity sows.

There were correlations for piglets’ birth weights and muscle weight (PB0.001; 0.73), muscle area (PB0.001; 0.54), and number of muscle fibers in the semitendinosus muscle (PB0.05; 0.39). These results show that the greater weight of piglets at birth was mainly due to semitendinosus muscle weight and secondarily to muscle area, and not to semitendinosus muscle fiber number. However, an influence of piglets’ weight class on the number of semitendinosus secondary muscle fibers (Table 3) was also found, where light-weight piglets had lower numbers of semitendinosus secondary muscle fibers when compared with heavier piglets.

This result is in agreement with many authors (Powell and Aberle 1980, 1981; Wigmore and Stickland 1983; Handel and Stickland 1987; Foxcroft et al. 2006; Trista´n et al. 2009) who observed that light-weight piglets had a lower total number of muscle fibers due to a lower number of secondary muscle fibers, compared with heavy piglets. Nissen et al. (2004) found greater amounts of DNA and RNA per fiber in high-weight pigs than in low-weight pigs and suggested that this supports an increased protein turnover.

Table 3. Influence of piglet’s weight class on piglets’ number of semitendinosus muscle fibers.

Differences in muscle characteristics of piglets related to the sow parity - Image 5

^{a, b} Within a column, means without a common letter differ (PB0.05).

Moreover, Rehfeldt and Kuhn (2006) observed lower muscular protein concentration and activity of creatine kinase (a marker of muscular differentiation) in lowweight than in high-weight piglets.

These results suggest that the higher number of fetuses in high parity sows, which can mimic intrauterine growth retardation, affects fetal development by reducing the average birth weight, due at least in part by impairing muscle accretion, as suggested by Alvarenga et al. (2013). Even with a low correlation between birth weight and the number of secondary fibers in the muscle, these fibers are important during fetal development, since impairment in prenatal cell proliferation, differentiation, and protein accretion in secondary muscle fibers leads to low-weight piglets (Rehfeldt and Kuhn 2006).

Conclusions

The present study showed that piglets’ birth weights were affected by muscle characteristics. However, the birth weight had the strongest correlation with muscle weight and not with secondary muscle fiber number, as it was hypothesized. These results also indicate that parity would be an important factor influencing fetus development in terms of myofibers.

1. Alvarenga, A. L. N., Chiarini-Garcia, H., Cardeal, P. C., Moreira, L. P., Foxcroft, G. R., Fontes, D. O. and Almeida, F. R. C. L. 2013. Intra-uterine growth retardation affects birthweight and postnatal development in pigs, impairing muscle accretion, duodenal mucosa morphology and carcass traits. Reprod. Fert. Devel. 25: 387_395.

2. Ashworth, C. J., Finch, J. M., Page, K. R., Nwagwu, M. O. and Mcardle, H. J. 2001. Causes and consequences of fetal growth retardation in pigs. Reproduction 58: 233_246.

3. Ashmore, C. R., Addis, P. B. and Loerr, L. 1973. Development of muscle fibers in the fetal pig. J. Anim. Sci. 36: 1088_1093.

4. Bee, G. 2004. Effect of early gestation feeding, birth weight, and gender of progeny on muscle fiber characteristics of pigs at slaughter. J. Anim. Sci. 82: 826_836.

5. Cavalcante-Neto, A., Tholon, P., Lui, J. F., Lara, M. A. C., Fonseca, C., Ribeiro, M. N. and Sarmento, J. L. R. 2011. Random regression models with different residual variance structures for describing litter size in swine. Cieˆnc. Agro. 42: 1043_1050.

6. Cerisuelo, A., Baucells, M. D., Gasa, J., Coma, J., Carrio´ n, D., Chapinal, N. and Sala, R. 2009. Increased sow nutrition during midgestation affects muscle fiber development and meat quality, with no consequences on growth performance. J. Anim. Sci. 87: 729_739.

7. Dwyer, C. M. and Stickland, N. C. 1991. Sources of variation in myofibre number within and between litters of pigs. Anim. Prod. 52: 527_533.

8. Dwyer, C. M., Fletcher, J. M. and Stickland, N. C. 1994. The influence of maternal nutrition on muscle fiber number development in the porcine fetus and on subsequent postnatal growth. J. Anim. Sci. 72: 911_917.

9. Eissen, J. J., Kanis, E. and Kemp, B. 2000. Sows factors affecting voluntary feed intake during lactation. Livest. Prod. Sci. 64: 147_165.

10. Ender, K. 1994. Future demands on meat quality. Arch. Anim. Breed. 37: 137_142.

11. Ferguson, E. M., Slevin, J., Edwards, S. A., Hunter, M. G. and Ashworth, C. J. 2006. Effect of alterations in the quantity and composition of the pre-mating diet on embryo survival and foetal growth in the pig. Anim. Reprod. Sci. 96: 89_103.

12. Foxcroft, G. R., Dixon, W. T., Novak, S., Putman, C. T., Town, S. C. and Vinsky, M. D. 2006. The biological basis for prenatal programming of postnatal performance in pigs. J. Anim. Sci. 84: 105_112.

13. Foxcroft, G. R. and Town, S. 2004. Prenatal programming of postnatal performance _ The unseen cause of variance. Adv. Pork Prod. 5: 269_279.

14. Gatford, K. L., Ekert, J. E., Blackmore, K., De Blasio, M. J., Boyce, J. M., Owens, J. A., Campbell, R. G. and Owens, P. C. 2003. Variable maternal nutrition and growth hormone treatment in the second quarter of pregnancy in pigs alter semitendinosus muscle in adolescent progeny. J. Nutr. 90: 283_293.

15. Handel, S. E. and Stickland, N. C. 1987. The growth and differentiation of porcine sckeletal muscle fibers types and the influence of birthweight. J. Anat. Vet. 152: 107_119.

16. Holanda, M. C. R., Barbosa, S. B. P., Sampaio, I. B. M., Santos, E. S. and Santoro, K. R. 2005. Litter size and average weights at birth and at 21 days of age of Large White piglets. Arq. Bras. Med. Vet. Zoo. 57: 539_544.

17. Karunaratne, J., Bayol, S., Ashton, C. and Stickland, N. 2007. Potential mechanisms for the nutritional control of muscle composition in developing pigs. Arch. Tierz. 50: 5.

18. Kelly, A. M. and Zacks, S. I. 1969. The histogenesis of rat intercostals muscle. J. Cell Biol. 42: 135_153.

19. Larzul, C., Lefaucheur, L., Ecolan, P., Gogue, J., Talmant, A., Sellier, P., Roy, P. I., Monin, G. and Le, R. P. 1997. Phenotypic and genetic parameters for longissimus muscle fiber characteristics in relation to growth, carcass, and meat quality traits in Large White pigs. J. Anim. Sci. 75: 3126_3137.

20. Littell, R. C., Milliken, G. A., Stroup, W. W. and Wolfinger, R. D. 1996. SAS system for mixed models. SAS Institute, Inc., Cary, NC.

21. Mahan, D. C. and Peters, J. C. 2004. Long-term effects of dietary organic and inorganic selenium sources and levels on reproducing sows and their progeny. J. Anim. Sci. 82: 1343_1358.

22. Miller, L. R., Garwood, V. A. and Judge, M. D. 1975. Factors affected porcine muscle fiber type, diameter and number. J. Anim. Sci. 41: 66_77.

23. Musser, R. E., Davis, D. L., Dritz, S. S., Tokach, M. D., Nelssen, J. L., Minton, J. E. and Goodband, R. D. 2004. Conceptus and maternal responses to increased feed intake during early gestation in pigs. J. Anim. Sci. 82: 3154_3161.

24. Nissen, P. M., Danielsen, V. O., Jorgensen, P. F. and Oksbjerg, N. 2003. Increased maternal nutrition of sows has no beneficial effects on muscle fiber number or postnatal growth and has no impact on the meat quality of the offspring. J. Anim. Sci. 81: 3018_3027.

25. Nissen, P. M., Jorgensen, P. F. and Oksbjerg, N. 2004. Withinlitter variation in muscle fiber characteristics, pig performance, and meat quality traits. J. Anim. Sci. 82: 414_421.

26. Powell, S. E. and Aberle, E. D. 1980. Effects of birth weight on growth and carcass composition of swine. J. Anim. Sci. 50: 860_868.

27. Powell, S. E. and Aberle, E. D. 1981. Skeletal muscle and adipose tissue cellularity in runt and normal birth weight swine. J. Anim. Sci. 52: 748_756.

28. Rehfeldt, C., Kuhn, G., Vanselow, J., Furbass, R., Fiedler, I., Nurnberg, G., Clelland, A. K., Stickland, N. C. and Ender, K. 2001. Maternal treatment with somatotropin during early gestation affects basic events of myogenesis in pigs. Cell Tissue Res. 306: 429_440.

29. Rehfeldt, C. and Kunh, G. 2006. Consequences of birth weight for postnatal growth performance and carcass quality in pigs as related to myogenesis. J. Anim. Sci. 84: 113_123.

30. Rehfeldt, C., Lefaucheur, L., Block, J., Stabenow, B., Pfuhl, R., Otten, W., Metges, C. C. and Kalbe, C. 2012. Limited and excess protein intake of pregnant gilts differently affects body composition and cellularity of skeletal muscle and subcutaneous adipose tissue of newborn and weanling piglets. Eur. J. Nutr. 51: 151_165.

31. Reynolds, L. P., Borowicz, P. P., Vonnahme, K. A., Johnson, M. L., Grazul-Bilska, A. T., Wallace, J. M., Caton, J. S. and Redmer, D. A. 2005. Animal models of placental angiogenesis. Placenta 26: 689_708.

32. Solomon, M. B., Campbell, R. G. and Steele, N. C. 1990. Effect of sex and exogenous porcine somatotropin on longissimus muscle fiber characteristics of growing pigs. J. Anim. Sci. 68: 1176_1181.

33. Sosnicki, A. 1987. Association of micrometric traits on meat quality, fattening and slaughter traits in the pig. J. Anim. Sci. 64: 1412_1418.

34. Town, S., Putman, C., Turchinsky, J., Dixon, W. T. and Foxcroft, G. R. 2004. Number of conceptuses in utero affects porcine fetal muscle development. Reproduction 128: 443_454. Trista´ n, F., Rivero, M. A., Albors, O. L., Ramis, G., Va´ squez, J. M., Marti´nez, M., Marti´nez, J. S. and Gil, F. 2009. Relationship of birth weight with the size, number and proportion of fibres in the pig semitendinosus muscle. Anat. Hist. Embryo 38: 275_278.

35. Wigmore, P. M. C. and Stickland, N. C. 1983. Muscle development in large and small pigs fetuses. J. Anat. 137: 235_245.

36. Zhu, M. J., Han, B., Tong, J., Ma, C., Kimzey, J. M., Underwood, K. R., Xiao, Y., Hess, B. W., Ford, S. P.,

37. Nathanielsz, P. W. and Du, M. 2008. AMP-activated protein kinase signalling pathways are down regulated and skeletal muscle development impaired in fetuses of obese, overnourished sheep. J. Physiol. 586: 2651_2664.