Viscera Characteristics of MSTN-Edited Heterozygous Pigs

Myostatin (MSTN) is a protein that negatively regulates growth of skeletal muscle, and inactivation of MSTN improves the mass of skeletal muscle. Our previous work found that MSTN +/− pigs have higher muscle depth and lower fat depth compared to wild type without any developmental problems. Therefore, MSTN-edited pigs are most likely to appear as heterozygotes in the potential future market, but the characteristics of organs in digestive and reproductive system of pigs with MSTN gene editing remains unclear. Here, we investigated the histological of the organs in the digestive system and reproductive system in MSTN gene heterozygotes at adult stages. The length of intestine was further compared between adult heterozygous and wild type pigs. We found no significant differences in histomorphology of organs, including heart, duodenum, jejunum, ileum, cecum, colon, testis, epididymis, ovaries, oviducts and uterus, between individuals from two genotypes. Moreover, there was no significant difference in the average length of intestine in adult pigs. Our data provide a reference for further clarifying the applications of MSTN gene edited pigs.

Since pigs are farmed as a major source of animal protein for humans, generation of pigs with MSTN loss-of-function mutations has become a research priority in order to obtain breeding stock with better meat quality and higher economic value. Previous studies have reported the successful production of healthy MSTN gene-inactivated Chinese Meishan pigs (Qian et al., 2015) and Erhualian pigs (Wang et al., 2017). In addition to phenotypic changes to muscle and fat tissues, organs weights were generally significantly reduced among MSTN −/− animals compared with the organs in their wild type (WT) counterparts (Bünger et al., 2004;Fiems, 2012;Luo et al., 2019). Bünger (2004) found that in MSTN knockout mice, the significant increase in muscle weight and decrease in body fat content were accompanied by significant reductions in the weights of heart, liver, kidney and digestive tract (Bünger et al., 2004). Luo (2019) examined the organ weights of MSTN −/− , MSTN +/− and WT piglets and found that the visceral weight of MSTN −/− homozygous piglets was significantly lower than that of MSTN +/− heterozygotes and WT piglets (Luo et al., 2019). These studies show that animals with double site knockout of MSTN have lighter internal organs than WT individuals. The development of organs has a direct impact on an individual's health, and the weights of body tissues and organs directly affect the slaughter rate, an important index of pig slaughtering performance, which in turn affects economic benefits. However, the mechanism of how MSTN regulates visceral development remains unclear. These findings all raise concerns about the health and welfare of MSTN edited animals.
In our previous study, MSTN +/− Large White (LW) pigs underwent exon 3 editing were prepared. The lean meat production of the MSTN +/− LW pigs were significantly higher than that of the WT (75.50%: 69.44%, p < 0.0001). Conversely, the fat meat rates of MSTN +/− pigs were significantly lower than that of the WT pigs (5.11%: 10.36%, p = 0.0022). The majority of amino acids content in the MSTN-edited LW pork were significantly higher than that in the WT pork; and the levels of polyunsaturated fatty acids were enriched in the MSTN-edited LW lean meat (Fan et al., 2021). Pigs with a naturally occurring single copy MSTN mutation have higher muscle depth and lower fat depth compared to the WT (Matika et al., 2019). Therefore, the heterozygous of MSTN gene-edited pigs have excellent production performance and are likely to be used in market applications in the future. However, there has been no systematic study on the digestive tract organs and reproductive organs of MSTN +/− pigs. The structure and function of intestine can affect nutrient absorption from feed. And the function of reproductive system organs determines the reproductive efficiency of the breeding herd. These are important factors to the economic benefit of pig production. Therefore, we examined the histological features of major organs in digestive and reproductive system in adults, and also contrasted the intestine length of MSTN +/− LW pigs and their WT half-sibs. This work will provide an important reference for MSTN gene edited pigs that can be used in future pig breeding.

Animals
The pigs had ad libitum access to a commercial pig diet and water throughout the experimental period. All animal studies were approved by the Animal Welfare and Research Ethics Committee at the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences. Here, we used newborn, 5month-old and 8-month-old MSTN +/− and WT pigs to study the viscera development characteristics.

Histological Analysis
Paraffin-embedded sections (4 μm thick) of various organs of MSTN +/− , and WT piglets, 5-month-old pigs, and 8-month-old pigs were made using a histotome. The slices were dewaxed, hydrated with gradient alcohol, stained with hematoxylin solution for 15 min, counterstained with 0.5% eosin solution for 5 min, dehydrated with gradient alcohol, cleared, and sealed (Feldman and Wolfe, 2014). After staining, images were acquired using a LEICA DMi8 microscope. Then, we assessed which cell types were in the images and the morphological characteristics of the cells to detect whether there were any changes in the tissue structure between the two genotypes.

Characterization and Analysis of Organs
In total, 17 MSTN +/− and 18 WT LW pigs were slaughtered at 8 months old, and the length of intestinal length was measured as follows: Small intestinal length (m): Length from the pylorus to the ileocecal junction.
Large intestine length (m): The length from the ileocecal junction to the anus.

Statistical Analysis
Data were analyzed using SPSS 19 software (SPSS, Inc., Chicago, IL, United States). All data were expressed as Mean ± SEM. The independent sample t-test (Student's t-test) was used to calculate the significance, and p < 0.05 was used as the criterion for significance.

No Significant Changes in the Histomorphology of Heart Between WT and MSTN +/− Pigs
The formalin-fixed paraffin-embedded heart tissue of MSTN +/− and WT piglets were stained by Hematoxylin and Eosin (H&E) for histomorphological comparison. Results showed that the myocardial fibers of newborn WT and MSTN pigs were both short and cylindrical, branched and interconnected in a network, and there were intercalated discs at the junction between adjacent myocardial fibers ( Figures 1A,B). Furthermore, we compared the sizes and weights of heart of 17 MSTN +/− and 18 WT 8-monthold pigs. The appearance and sizes of heart did not markedly differ between the MSTN +/− and WT adult pigs ( Figure 1C). There was also, no significant difference of average heart weight between MSTN +/− (406.5 ± 15 g) and WT (452.3 ± 32.4 g).

No Significant Differences in Histomorphology of Major Organs in Digestive System Between Individuals From Two Genotypes at 5-month-Old
When pigs were 5 months old, we carried out histomorphological examination of the duodenum, jejunum, ileum, cecum, and colon. The structure of duodenum, jejunum and ileum of pigs was intact, and the small intestinal villi were slender and orderly arranged, the morphology and structure of epithelial cells were intact and clear, the cup cells were evenly distributed and in large number, the villi height was normal, the morphology and structure of crypt were clear, and the depth of crypt was normal (Figures 2A-C). The colonic structure was intact. The epithelial cells of the mucosa are arranged neatly and clearly defined, and the boundary between the epithelial layer and lamina propria is clear. The columnar cells are tall and the nuclei are elliptic, and the goblet cells are goblet shaped near the base of the cells ( Figure 3A). The structure of the cecum is complete and closely arranged as that of WT pigs. The villi are orderly, uniform in length and very dense ( Figure 3B). Furthermore, we compared the intestinal length of 17 MSTN +/− and 18 WT 8-month-old pigs. No any significant difference between genotypes in total intestinal length (combined length of small and large intestines, Table 1). According to the results, there were no significant morphological and length differences between MSTN +/− and WT in these tissues.

No Significant Changes in the Reproductive Organs Between Adult WT and MSTN +/− Pigs at 8-month-Old
Reproductive organs, including testis, epididymis, ovaries, oviducts and uterus, were collected from pigs aged 8 months. And these tissues were processed for H&E staining. The MSTN +/− pigs showed normal histology of testis and epididymides, and sperm were present in testis and epididymides (Figure 4). Different follicular stages and atretic follicles existed in ovarian sections ( Figure 5A). Compared with the WT group, histological analysis under light microscopy showed no morphologic differences in the ovaries, oviducts, and uterus histology of the MSTN group ( Figure 5).

DISCUSSION
Many laboratories have encountered problems at different stages in the process of MSTN deletion or editing. For example, MSTN-  knockout Large White/Landrace×Duroc piglets died within 24 h after birth, and weighed less at birth than WT piglets. The single surviving piglet was sacrificed at 29 days old because of weakness in its hindlimbs (Rao et al., 2016). Similarly, eight of the MSTN −/− Landrace newborns generated with CRISPR/Cas9 editing died within a week . Pigs with naturally occurring MSTN mutations were reported in 2019 (Matika et al., 2019). However, piglets that were homozygous for this mutant allele suffered from lameness syndrome and did not survive when growth with the live weight above 40 kg (Matika et al., 2019). MSTN homozygous knockout in LW pigs leads to lameness, in agreement with other studies. Subsequently, pigs used for this study carrying single, edited copies of MSTN were propagated to expand the population for up to 8 years, which was sufficient to show that these MSTN +/− swine exhibited no developmental defects related to the mutant allele. MSTN +/− heterozygotes had a significant decrease in fat content that was accompanied by an increase in lean meat content (Fan et al., 2021). Therefore, MSTN-edited pigs are most likely to appear as heterozygotes in the potential future market.
Cardiac performance, reserve, and capability of the smaller heart are reduced in MSTN −/− animals compared to WT (Amory et al., 1993). The lower cardiac performance of MSTN −/− animals leading more quickly to exhaustion after severe exercise (Monin et al., 1974), which may even terminate in sudden death (Holmes et al., 1973). Our results showed that there were no histological abnormalities of cardiac muscle structures, and no significant difference in weight between individuals from two genotypes adults. MSTN +/− LW pigs did not suffer from reduced heart function, so there is no need to take extra precautions for animals.
It is well known that the small intestine consists of duodenum, jejunum, and ileum, is responsible for digestion and absorption of nutrients, water, and electrolytes, while the large intestine dividing into cecum and colon, is responsible to absorb water. Notably, the digestive disorders are related to intestinal dysfunction (Johnson, 1985;Soybel, 2005;Iji, 2008). Besides, the length of the digestive tract is positively correlated with nutrient absorption, while the decreases in the digestive tract length may limit the basic metabolism and nutrient availability of  the body (Ortigues and Doreau, 1995;Greig et al., 2019). As reported by several authors, a consequence of the smaller digestive tract is reduced feed intake capacity (Clinquart et al., 1995;Fiems et al., 1997). The decrease in feed intake means that MSTN −/− animals do not efficiently utilize low quality feed, and the costs of rearing will increase. According to our results, the histological structure and length of intestinal in MSTN +/− swine were not affected by single copy MSTN mutation. This coincides with our previous finding that no differences were detected in feed conversion ratio values between MSTN +/− and WT LW (Fan et al., 2021). Hence, there were no differences between MSTN +/− and WT LW pigs in the function of digestive tract. Spermatogenesis occurs in the testis and completes in the epididymis (de Kretser et al., 1998). Meanwhile, the ovary, oviduct and uterus are critical organs responsible for the oogenesis and embryonic development (Bagnell et al., 2017). The BBDM cows have significant longer calving interval than dairy cows (averaging 435 days compared to 393 days) (Hanzen et al., 1994). That may be caused by underdeveloped maternal reproductive tract and increased birth weight of calves, which make cesarean section an elective operation (Kolkman et al., 2007). A longer calving interval can be affected by the cesarean section, which significantly reduces the subsequent pregnancy rate (Patterson et al., 1981), but this is not the sole cause. Compared with Holstein cows, BBDM cows have significantly poorer semen quality (Hoflack et al., 2006). Nevertheless, BBDM cows have better egg quality than Holstein cows (Leroy et al., 2005). Other studies have shown that compared with the control group, MSTN-immunized mice had reduced litter number, but normal embryo development in both groups (Liang et al., 2007). These results are consistent with those obtained with BBDM (Leroy et al., 2005). This indicates that the completely loss of MSTN function does not affect the function of the uterus, but  reduces the quality of sperm and increases litter birth weight. Our results previous work showed that the total number born, the number born alive and litter birth weight of the MSTN +/− piglets did not differ from those of the WT (Fan et al., 2021). Here, results showed that there was no significant difference in the histomorphology of testis, epididymis, ovaries, oviducts and uterus in MSTN +/− LW compared with WT pigs. Additionally, our MSTN +/− LW pigs are now in their eight generation (Fan et al., 2021). Therefore, it can be seen that the characteristics of reproductive organs of MSTN +/− LW pigs are similar to WT pigs.

CONCLUSION
In summary, MSTN +/− LW pigs had no significant differences in the histomorphology of the heart, duodenum, jejunum, ileum, cecum, or colon, nor in the lengths of the large and small intestine compared to WT pigs. Similarly, no morphologic abnormality was evident in testis, epididymis, ovaries, oviducts and uterus. These results indicated that the characteristics of heart, digestive and reproductive organs of MSTN +/− heterozygotes are similar to that of WT LW pigs.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Welfare and Research Ethics Committee at the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (IAS2012-11). The pigs used in this paper were prepared by our team.

AUTHOR CONTRIBUTIONS
KL, ZF, and YP designed and managed the project. YP, ZF, YS, and CC analyzed the data and performed all animal works and collected biological samples. YP wrote the manuscript. ZF, YM, BL, HL, and KL revised the paper. All authors approved the final version of the manuscript.