Investigation on the Growth and Development of Muscle Fibers in Black Goat Lambs

Lin ZHANG Shengxia BAI Quzhe EMU Feng XU Jingsheng FAN Xuemei LI Bo LI Bin ZHANG Chunmei LI Chaorui XIONG

Abstract   [Objectives]  This study was conducted to analyze and study the growth and development of muscle fibers at different stages， so as to provide a theoretical basis for subsequent research on developmental biology and nutritional regulation.  [Methods]  The body weights of black goat lambs in groups at different month age （0-6 months old） were recorded， and the diameter， area and number of muscle fibers were measured by tissue section and hematoxylin-eosin staining. The curves of body weight and muscle fiber growth and development of black goat lambs were drawn.  [Results]  The black goat lambs maintained a high absolute growth intensity of 2.95-4.35 kg/30 d throughout the rearing cycle， and the body weight continued to increase significantly， while the relative growth coefficient decreased from 93.05% at the age of 30 d to 13.83% at the age of 180 d. The diameter and area of muscle fibers in black goat lambs showed obvious "three-stage" growth and development characteristics， and showed different trends of gender advantages in different stages.  [Conclusions]  This study will help to further optimize the key supporting technologies for production and promote the diversified and comprehensive development of the mutton sheep industry in Sichuan Province.

Key words  Black goat lamb; Muscle fiber; Growth and development; Law 

The mutton sheep industry occupies an important position in Sichuan’s animal husbandry， and the stock of meat-type goats， the output of mutton， and the resources of breeds all rank in the forefront of China. With the change of market consumption trends， consumers have higher and higher requirements for mutton quality， especially mutton tenderness. In response to these changes，  Sichuan  Academy of Animal Husbandry Science bred a new breed of black goat lamb with fast growth rate， high reproductive performance and high carcass meat yield using southern Sichuan black goats （Zigong type） as the breeding material， and formed a comprehensive breeding system for black goat lambs based on the optimal "fine+coarse" feed ratio  [1-2] . The black goat lambs have the characteristics of low fattening cost， excellent meat quality， fast growth rate in the early stage， high utilization rate of forage， less pressure on the environment， and significant yield  [2] . Because of its unique meat flavor， tenderness and juiciness， finished lamb mutton is deeply loved by consumers and has a large market demand. Therefore， the safe and efficient production of finished lamb mutton has become an important model for the structural transformation and industrial upgrading of the mutton sheep industry in Sichuan Province  [1] .

The realization of the key supporting technologies for the  production  of black goat lambs is based on the rapid growth and development of muscle fibers. The diameter， density and area of muscle fibers are important indices reflecting muscle quality  [3] . Generally， the diameter of muscle fibers in livestock and poultry is about 10-100 μm. The diameter of muscle fibers determines the density of muscle fibers and is inversely related to muscle tenderness  [4] . Wang  et al.   [5]  studied the diameter and density of muscle fibers in the same muscle of Hu sheep at different ages. The results showed that the age of Hu sheep was also inversely correlated with muscle tenderness， and muscle tenderness gradually deteriorated with age. Yang  et al.   [6]  found that as the age of Danba Procapra gutturosa and Tibetan goats， the body weight， the diameter of muscle fibers and the cross-sectional area increased， while the density decreased， and the tenderness of muscles decreased. However， there have been no reports on the change laws of muscle fibers in black goat lambs. In this study， the change laws of muscle fibers in the whole production process of finished lambs of black goat lambs were revealed， aiming to provide a basic theory for further optimizing the breeding and production of black goat lambs.

Materials and Methods 

Samples and reagents

Samples： The black goat lamb test was carried out in Rong County， Zigong City， Sichuan Province and Shigangzhai Sheep Farm of Mushanyang Breeding Professional Cooperative. The test was implemented in May 2021. Under the same growth environment conditions， three black goat lambs， male and female， at the ages of 3， 30， 60， 90， 120， 150， and 180 d， were taken， respectively， and their body weights were recorded. After slaughter， the longissimus dorsi muscle of 2 cm×2 cm×0.5 cm was taken and fixed with 10% neutral formaldehyde.

Reagents： 10% neutral formaldehyde fixing solution， 70%， 80% and 90% ethanol solutions， clear xylene reagent， hematoxylin staining solution， eosin staining solution， hydrochloric acid-ethanol differentiation solution， and neutral gum sealant.

Instruments and equipment

Histotome （Leica-2016， Germany）; TSJ-II tissue dehydrator， BMJ-III type embedding machine， PHY-III water bath-slide drier （Changzhou Zhongwei Electronic Instrument Co.， Ltd.）; digital trinocular camera microscope （BA210Digital， Motic China Group Co.， Ltd.）.

Tissue sectioning and hematoxylin-eosin staining

Dehydration procedures for fixed tissue included： 4 h in 75% ethanol for， 2 h in 85% ethanol， 1 h in 95% ethanol， 0.5 h in 100% ethanol， 0.5 h in 100% ethanol， 0.5 h in 100% ethanol， 0.5 h in 100% ethanol， 10 min in xylene， 10 min in xylene， 1 h in paraffin， 2 h in paraffin， 3 h in paraffin. After embedding and sectioning， following procedures were performed： dewaxing to water， staining with hematoxylin for 10-20 min， rinsing with tap water for 1-3 min， differentiating in hydrochloric acid-ethanol for 5-10 s， rinsing with tap water for 1-3 min， incubating in warm water at 50 ℃ until the appearance of blue， rinsing with tap water for 1-3 min， putting in 85% ethanol for 3-5 min， staining with eosin for 3-5 min， washing with water for 3-5 s， dehydrating with gradient ethanol， transparentizing in xylene， and sealing with neutral gum.

Image and data acquisition

The tissue section images were obtained by the BA210Digital digital microscope camera system. Three 400x images were  collected  on the better area in each section， and the images were imported into Motic Images Advanced for data collection. The measurement indices included muscle fiber diameter and muscle fiber  area. Meanwhile， the number of muscle fibers was counted in each image.

Growth factor analysis

Absolute growth coefficient：  G= W2-W1 t2-t1

Relative growth coefficient：  R= W2-W1  W2+W1 2   ×100%

In the formulas，  Wi  represents the body weight at each measurement time point， and  ti  represents each measurement time point.

Statistical Analysis

The test results were expressed as means±standard error. Student’s  t  test， one-way ANOVA and Tukey’s test were adopted to conduct multiple comparisons and significance analysis of the data between various time points. When  P <0.05， the difference was considered significant.

Results and Analysis 

Morphological observation of longissimus dorsi muscle sections from black goat lambs

The results of the study showed that the longissimus dorsi muscle of 3-day-old lambs had mature muscle fibers， and dozens of muscle fibers gathered together to form primary muscle bundles. Muscle fibers were stained with hematoxylin-eosin to show pink myofibers and blue myocyte nuclei distributed on the inner side of the myofiber cell membrane. It can be seen from Fig. 1 that with the increase of age， the cross-sectional area of muscle fibers gradually increased， and the number of muscle fibers in each visual field gradually decreased.

Body weight and muscle fiber growth and development of black goat lambs

During the 180-day rearing period， the black goat lambs maintained a high growth rate （Fig. 2）， and the absolute growth coefficient remained between 2.95-4.35 kg/30 d （Table 1）， especially in the first 60 d of rearing period， during which the weight gain was maintained at more than 4 kg every 30 d. During the following 120-d rearing period， the weight gain was maintained at  about  3 kg every 30 d. The growth intensity of the black goat lambs changed greatly， and its relative growth coefficient decreased from 93.05% to 13.83%， especially in the first 90 d， during which the reduction was as high as 69.33%， while the relative growth coefficient from 120 to 180 d remained below 20%. On the one hand， the relative growth coefficient was relatively large in the first two months due to the light weight and small base at birth， and the values were 93.05% and 46.77%， respectively; and on the other hand， the rearing period of the black goat lambs includes the first 60 d of lactation and the last 120 d of growth， which were two rearing periods with different nutrition levels， having more obvious effects on the body weight growth characteristics and the diameter and area of muscle fibers in black goat lambs.

Agricultural Biotechnology 2022

Tukey’s multiple comparisons were performed on the body weights of black goat lambs at 7 time points， and only the body weights of the three adjacent time points from 3 to 60 d showed significant pairwise differences （ P <0.05）. From 60 to 180 d， the body weights of black goat lambs at the two time points separated by 60 d were significantly different （ P <0.05）. It can be seen from Fig. 2 that the growth intensity of the black goat lambs in the period of 60-180 d was significantly lower than that in the period of 3-60 d.

Compared with the continuous increase in body weight， the growth of muscle fiber diameter and area in the black goat lambs was divided into three stages （Fig. 3 and Fig. 4）： 3-60 d of  lactation，  60-120 d of nursery and 120-180 d of growth. In  lactation  and growth periods， both the diameter and area of muscle fibers increased significantly during the 60 d rearing period  （ P <0.05） ， while during the 60-120 d nursery period， the diameter and area of muscle fibers remained stable and did not show significant growth  （ P >0.05）.  The correlation analysis showed that there was an extremely significant positive correlation between the diameter and area of muscle fibers，  R  2=0.945 （ P <0.001）. In addition， we counted the number of muscle fibers per unit （mm 2）. It can be seen from Fig. 5 that the number of muscle fibers per unit area gradually decreased （ P <0.001） throughout the rearing period of the black goat lambs， but its changes did not show obvious three-stage characteristics. Correlation analysis showed that the number of muscle fibers per unit area was significantly negatively correlated with muscle fiber area，  R  2=0.677 3 （ P <0.001）.

Effects of gender on body weight change and muscle fiber growth in black goat lambs

On the basis of the above studies， we also compared the effects of different genders on the growth and muscle fiber development of the black goat lambs. It can be seen from Fig. 6 that the body weight of 150-day-old rams was significantly greater than that of ewes （ P <0.05）. From Fig. 7， it can be seen that the diameter of muscle fibers of 3-day-old ewes was significantly larger than that of rams （ P <0.05）. From Fig. 8， it can be seen that the muscle fiber area of 30-day-old rams was significantly larger than that of  ewes  （ P <0.05）. As can be seen from Fig. 9， the number of muscle fibers per square milimeter of 120-day-old ewes was significantly less than that of rams （ P <0.05）. During the 180-day rearing period， the body weight accumulation curve of ewes was consistently lower than that of rams （Fig. 6）， but for muscle fiber diameter （Fig. 7）， muscle fiber area （Fig. 8）， and number of muscle fibers per unit area （Fig. 9）， ewes showed a trend of overtaking rams between 60 and 120 d of age， and the number of muscle fibers per unit area at 120 d of age showed a significant difference （ P <0.05）.

Discussion and Conclusions

In this study， the growth and development and muscle fiber changes of the black goat lambs were systematically studied by tracking the body weight and muscle fiber changes of the black goat lambs， and the change laws of body weight and muscle fibers in the black goat lambs were revealed.

The results showed that the absolute growth coefficient of the black goat lambs before the age of 180 d was maintained at more than 3 kg per 30 d， and they maintained a high growth intensity throughout the rearing period  [7] . Compared with the relatively stable weight gain， the change curves of muscle fiber diameter and muscle fiber area of the black goat lambs showed the characteristics of three-stage growth including the lactation period， nursery period and growth period. One-year-old Egyptian RAHMANI and OSSIMI lambs also show a three-stage growth pattern  [8] . It is mainly related to the production and feeding system of finished lambs. Lambs during lactation have the characteristics of high nutrient conversion rate and high growth intensity， while during the nursery period， weaning stress induces the full development of the rumen and other internal organs related to the digestive system in lambs to adapt to the transition of fodder feeding  [9] . Therefore， we guess that the body weight change of black goat lambs during the nursery period is mainly reflected in the development and maturity of the internal organs of lambs， supplemented by the development and growth of muscle tissue. During the growing period， lambs complete the transition from the lactation period to the fodder period， and continue to maintain high growth rates and restart muscle development.

In the three stages of muscle fiber growth and development， the black goat lambs showed different laws in different sexes of male and female. Among Sudanese goats， rams exhibit higher feed conversion ratios， body weights before slaughter， and carcass weights， while ewes have more fat deposits in carcasses  [10] . During lactation， the change curves of muscle fiber diameter and muscle fiber area in black goat lambs showed that rams were better than ewes， which is consistent with the lagging growth rate of ewes in the early stage of Australian white sheep compared with  rams  [11] . After the nursery period， ewes showed better adaptability.

During the growth period， the growth of ewes’ muscle fibers tended to overtake the growth of rams’ muscle fibers. However， due to the insufficient number of experimental samples， there was no statistical difference between rams and ewes， and future work needs to further explore on the basis of this study.

Through this study， we found the change laws of body weight and muscle fibers in the black goat lambs. The specific manifestations were as follows： the body weight of the black goat lambs showed high-intensity growth throughout the rearing period， while the diameter and area of muscle fibers showed obvious three-stage characteristics， and lambs of different genders showed different gender advantages in different stages. This study will help to understand the rapid growth law and cellular mechanism of black goat lambs， and provide a theoretical basis for further optimizing the key supporting technology system for goat lamb production.

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