Authors: Xiaoyu Zhao, Jia Liu, Jiechuan Xiao, Yuan Yun, Chunjie Bo, Yuxin Gao, Lishuang Song, Chunling Bai, Zhuying Wei, Li Zhang, Lei Yang, Guangpeng Li, Guanghua Su
Categories: Research, Myostatin, Gene editing, Mongolian cattle, Reproductive performance, Hematological and biochemical physiology, Progesterone, Metabolome
Source: BMC Veterinary Research
Authors: Xiaoyu Zhao, Jia Liu, Jiechuan Xiao, Yuan Yun, Chunjie Bo, Yuxin Gao, Lishuang Song, Chunling Bai, Zhuying Wei, Li Zhang, Lei Yang, Guangpeng Li, Guanghua Su
The extended sexual maturity and prolonged gestation period of cattle limit their economic viability and hinder the breeding of superior genotypes. Reproductive efficiency is a critical factor in evaluating the economic value of cattle. Myostatin (MSTN) is a negative regulator of skeletal muscle growth. When it mutates, it promotes muscle development, providing a new strategy for modern beef cattle breeding. Although previous studies on MSTN gene-edited animals mainly focused on the mechanism of muscle growth, studies evaluating their reproductive performance and pregnancy outcomes are still limited. This study compared MSTN gene-edited Mongolian cattle (MT) with wild-type Mongolian cattle (WT) and Luxi cattle to assess the effect of MSTN editing on reproductive efficiency. Key parameters include reproductive organ morphology, hormone levels, blood metabolic profiles, and metabolomic changes during different gestation stages.
The statistical analysis results of reproductive organs (n = 10) and reproductive data (n = 20) showed that, compared with the WT Mongolian cattle, the MT cattle did not differ significantly Studies have shown that blood metabolites in estrus rate, pregnancy rate, calf birth rate, as well as the size of the ovaries and uterus (P > 0.05). Moreover, the dystocia rate of MT Mongolian cattle is significantly lower than that of WT Mongolian cattle (5.1% vs. 10.5%). Compared to WT cattle, the calves of the MT Mongolian cattle were heavier (P = 2.56815E-08), and the reproductive performance of the Mongolian cattle was better than Luxi cattle, specifically manifested as higher pregnancy success rate (WT-M vs. WT-L, 92.4% vs. 85.9%, MT-M vs. MT-L, 90.5% vs. 83.1%) and lower dystocia rate. The results of untargeted LC-MS metabolomics indicated that at different pregnancy stages, the MT cattle showed enhanced glucose metabolism, reduced lipid synthesis, lower circulating essential amino acids, and reduced tryptophan synthesis (n = 6). The results of serum ELISA at different pregnancy periods showed that the levels of E2 decreased and the levels of P4 increased in MT cattle (n = 3). These changes showed a regular pattern throughout the pregnancy cycle.
These findings demonstrated that the MSTN gene editing did not compromise the reproductive performance of cattle, supporting its potential application in beef cattle breeding program.
The online version contains supplementary material available at 10.1186/s12917-025-05079-y.
The Myostatin (MSTN) gene acts as a negative regulator for skeletal muscle growth and development. When it undergoes mutations, it can affect the protein structure, causing the organism to exhibit a “double-muscled” phenotype. This phenotype is characterized by a significant increase in skeletal muscle mass and a reduction in adipose tissue mass. While using gene-editing technology to induce MSTN gene mutations is considered a potential way to improve the meat production characteristics of livestock, there are still significant knowledge gaps regarding its impact on the reproductive performance of cattle.
The main objective of this study is to compare the reproductive production data and the size of reproductive organs between CRISPR/Cas9-based MSTN-mutant Mongolian cattle and non-mutated control cattle, and to explore the influence of MSTN gene mutation on reproductive phenotypes. Additionally, we aim to examine whether there are significant differences in reproductive traits among different breeds of cows after MSTN gene editing by comparing the reproductive-related detection indicators of MSTN gene-mutated Luxi cattle and non-mutated Luxi cattle. This research will provide data support for verifying the feasibility of MSTN gene-editing technology in beef cattle breeding.
Currently, natural mutations of the MSTN gene have been observed in multiple breeds such as Belgian Blue Cattle [1], Piedmontese [1], Charolais [2], Limousin [3], Blonde d’Aquitaine [4], and German Gelbvieh [5], all of which exhibit muscle hypertrophy. However, MSTN gene mutations are also the genetic cause of the double-muscled phenotype in various cattle breeds [6]. A major concern is that these mutations can lead to an increase in the body size of offspring, raising the probability of dystocia. For example, in Charolais cows with MSTN gene mutations, the risk of dystocia and calf mortality significantly increased, possibly due to the reduction in pelvic area and the increase in fetal weight caused by MSTN gene editing.
Studies have shown that blood metabolites in mammals change at different stages of pregnancy, which in turn affect reproductive efficiency [7–10]. The maternal nutritional level during the early stage of pregnancy is crucial for maintaining the pregnancy. Malnutrition in the early stage of pregnancy can affect the maturation and development of oocytes, thus leading to a decrease in the conception rate [11]. During the later stage of pregnancy, as the birth weight of calves increases significantly, the cow’s demand for nutrients such as glucose increases sharply, and energy balance is maintained through increased lipid metabolism [8, 12]. The levels of urea nitrogen, total protein and albumin in serum are indicators for evaluating the protein intake of cows. When cows consume diets with high crude protein content, the concentration of urea nitrogen in their plasma increases significantly. However, during non-pregnant period, excessively high concentration of urea nitrogen can have toxic effects on gametes and embryos. Also, it can reduce the synthesis of prostaglandins and decrease the secretion of progesterone, thereby causing delayed ovulation and prolonged uterine recovery [13, 14]. Estradiol (E2) and progesterone (P4) are the main bioactive progestogens secreted by ovary and placenta, and are of great significance for successful maintenance of pregnancy [15]. E2, along with other hormones, regulates the estrus of non-pregnant cows. When cows are not pregnant, an increase in E2 concentration leads to an increase in the secretion of luteinizing hormone (LH). LH works synergistically with follicle-stimulating hormone (FSH) to promote the growth and maturation of follicles. Moreover, during the late pregnancy stage, the secretion of E2 can act on the uterine smooth muscle, thereby enhancing the sensitivity of the uterine smooth muscle to oxytocin and strengthening uterine contractions to ensure a smooth delivery of the cows [16]. The secretion of progesterone can promote the hyperplasia of the endometrium and enhance the development and function of glands. Higher progesterone concentration during the early pregnancy stage is more conducive to embryo implantation [17].
Cows are typically monotocous animals with long reproductive cycles, which limit production efficiency. The Mongolian cattle, a native Chinese breed mainly found in Inner Mongolia and desert areas, has never been used for draft purposes. It is small-sized, slow-growing, and has low meat production. However, it is stress-resistant, disease-resistant, drought-resistant, and cold-resistant, and has high reproductive traits, making it an excellent breed for beef cattle farming [18].
The reproductive data of WT and MT Mongolian cattle were statistically analyzed, and the data of WT and MT Luxi cattle can be found in the study by Zhao et al. [17]. The results are shown in Table 1. There were no significant differences in the estrus rate, pregnancy rate, calf birth rate and survival rate between MT Mongolian cattle and WT Mongolian cattle (P >0.05). The calf survival rate of MT Mongolian cattle was slightly higher than that of WT Mongolian cattle, but there was no significant difference. In addition, the dystocia rate of MT Mongolian cattle was significantly lower than that of WT Mongolian cattle (P < 0.05). Compared with Luxi cattle, the Mongolian cattle exhibited better reproductive performance, specifically manifested as higher pregnancy rates of WT and MT Mongolian cattle, and their dystocia rates was lower than that of Luxi cattle.Table 1Statistics of reproductive indicators of WT and MT Mongolian cattle and Luxi cattleGroupSynchronous estrus quantityNumber of cow in estrus(%)Number of pregnant cows(%)Number of calves born(%)Number of difficult calving(%)Calf survival(%)WT-M7766(85.7)^a^61(92.4)^a^57(93.4)^a^6(10.5)^c^49(85.9)^a^MT-M11795(81.2)^a^86(90.5)^a^79(91.9)^a^4(5.1)^bc^75(94.9)^a^WT-L10185(84.2)^a^73(85.9)^b^67(91.7)^a^9(13.4)^ab^62(92.5)^a^MT-L11189(80.2)^a^74(83.1)^b^66(89.1)^a^12(18.2)^a^60(91.0)^a^In the same column, different letters indicate significant differences, while the same letters indicate no significant differences
To further analyze the reproductive performance of Mongolian cattle, we used an ultrasound machine to detect the status and size of the ovaries and uteruses. The results showed that the right ovaries of both Mongolian cattle and Luxi cattle were larger than the left ones, and this phenomenon was more obvious in MT cattle (Fig. 1A). Compared with WT and MT Luxi cattle, the right and left ovaries of Mongolian cattle were relatively smaller, which might be related to the smaller body size of Mongolian cattle (Fig. 1A). The measurement of uterine diameters showed that the MT Mongolian cattle was slightly larger than that of WT Mongolian cattle (1.7 ± 0.41 vs. 1.825 ± 0.15, P = 0.638), but the difference was not significant. The uterine diameters of MT Mongolian cattle were slightly larger than those of WT and MT Luxi cattle. The larger uterine diameter might be the reason for the lower dystocia rate of Mongolian cattle (Fig. 1B).Fig. 1Analysis of reproductive performance indicators of WT and MT Mongolian cattle and comparison of reproductive performance with Luxi cattle. A Statistics and comparison of the sizes of the left and right ovaries of WT and MT Mongolian cattle and Luxi cattle. B Analysis and comparison of the uterine diameters of WT and MT Mongolian cattle and Luxi cattle. C Statistics and comparison of the birth weights of calves of WT and MT Mongolian cattle and Luxi cattle. D Measurement and statistics of the pelvic height and pelvic width of WT and MT Mongolian cattle and Luxi cattle. E Statistical measurement of the pelvic area of WT and MT Mongolian cattle and Luxi cattle. F Statistics of the ratio of pelvic area to calf birth weight (pelvic-birth weight ratio) of WT and MT Mongolian cattle and Luxi cattle
Next, the pelvic dimensions of WT and MT Mongolian cattle were measured. The results showed that the pelvic height (19.65 ± 1.30 vs. 19.28 ± 0.88, P = 0.554), pelvic width (16.15 ± 1.58 vs. 15.36 ± 1.02, P = 0.290) and pelvic area (319.05 ± 51.26 vs. 296.93 ± 32.32, P = 0.357) of WT Mongolian cattle were all higher than those of MT Mongolian cattle, but the difference was not significant. Moreover, they were all slightly higher than those of Luxi cattle (Fig. 1D, E). Analysis of the birth weights of calves indicated that calves of MT Mongolian cattle had higher birth weights (32.14 ± 1.76 vs. 23.29 ± 2.23, P = 2.56815E-08), but the ratio of pelvic area to birth weight was significantly lower than that of WT Mongolian cattle (8.95 ± 0.66 vs. 13.63 ± 1.02, P = 4.9648E-08) (Fig. 1C, F). Compared with WT and MT Luxi cattle, the birth weights of calves of Mongolian cattle were slightly lower (Fig. 1C, F). These factors might explain the higher pregnancy rate and lower dystocia rate in Mongolian cattle.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{aligned} &\mathrm{estrus};\mathrm{rate}=\frac{\mathrm{number};\mathrm{of};\mathrm{cows};\mathrm{in};\mathrm{estrus}}{\mathrm{number};\mathrm{of};\mathrm{cows};\mathrm{in};\mathrm{estrus};\mathrm{during};\mathrm{the};\mathrm{same};\mathrm{period}}\times100%;\&\mathrm{pregnancy};\mathrm{rate}=\frac{\mathrm{number};\mathrm{of};\mathrm{pregnant};\mathrm{cows}}{\mathrm{number};\mathrm{of};\mathrm{cows};\mathrm{inseminated}}\times100% \end{aligned}
\usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \begin{aligned} &\mathrm{calving}\;\mathrm{rate}=\frac{\mathrm{number}\;\mathrm{of}\;\mathrm{calving}\;\mathrm{cows}}{\mathrm{number}\;\mathrm{of}\;\mathrm{pregnant}\;\mathrm{cows}}\times100\%\;\\&\mathrm{calving}\;\mathrm{success}\;\mathrm{rate}=\frac{\mathrm{number}\;\mathrm{of}\;\mathrm{cows}\;\mathrm{calving}\;\mathrm{normally}}{\mathrm{total}\;\mathrm{number}\;\mathrm{of}\;\mathrm{production}\;\mathrm{cows}}\times100\% \end{aligned} $$\end{document} ### Analysis of the differences in blood physiological and biochemical indicators between WT and MT Mongolian cattle at different gestation stages In order to further explore the reasons for the differences between WT and MT Mongolian cattle during pregnancy, we detected and analyzed the contents and changing trends of blood physiological and biochemical indicators at different pregnancy stages, and compared with Luxi cattle (the data of Luxi cattle are shown in the study by Zhao et al. [17]). The main physiological and biochemical indicators of blood include glucose metabolism indicators including glucose (Glu), lactic acid (LA) and lactate dehydrogenase (LDH), the lipid metabolism indicators including triglyceride (TG), cholesterol (CHO), high-density lipoprotein (HDL), and low-density lipoprotein (LDL), protein metabolism indicators including blood urea nitrogen (BUN), total protein (TP) and albumin (ALB)), and health-related indicators including aspartate aminotransferase (AST), creatinine, creatine kinase (CK) and bicarbonate (BC). The results were shown in Fig. 2, which illustrated the differences in blood glucose, blood lipid, protein metabolism, and health indicators of WT and MT Mongolian cattle at different stages of pregnancy, as well as the differences between WT and MT Mongolian cattle and WT and MT Luxi cattle.Fig. 2Analysis of blood physiological and biochemical indicators of WT and MT Mongolian cattle and Luxi cattle at different pregnancy stages. **A-C** Glucose metabolism indicators in the blood of WT and MT Mongolian cattle and Luxi cattle at different pregnancy stages. **A** Glucose levels in the blood of WT and MT Mongolian cattle and Luxi cattle. **B** Lactic acid levels in the blood of WT and MT Mongolian cattle and Luxi cattle. **C** LDH content in the blood of WT and MT Mongolian cattle and Luxi cattle. **D-G** Lipid metabolism indicators in the blood of WT and MT Mongolian cattle and Luxi cattle. **D** Detection and analysis of triglyceride content in the blood of WT and MT Mongolian cattle and Luxi cattle. **E** Cholesterol content in the blood of WT and MT Mongolian cattle and Luxi cattle. **F** HDL content in the blood of WT and MT Mongolian cattle and Luxi cattle. **G** LDL content in the blood of WT and MT Mongolian cattle and Luxi cattle. **H-J** Detection of protein metabolism indicators in the blood of WT and MT Mongolian cattle and Luxi cattle at different pregnancy stages. **H** BUN levels in the blood of WT and MT Mongolian cattle and Luxi cattle. **I** Total protein levels in the blood of WT and MT Mongolian cattle and Luxi cattle. **J** Albumin content in the blood of WT and MT Mongolian cattle and Luxi cattle. **K-N** Detection of health - related indicators in the blood of WT and MT Mongolian cattle and Luxi cattle at different pregnancy stages. **K** AST content in the blood of WT and MT Mongolian cattle and Luxi cattle. **L** Creatinine levels in the blood of WT and MT Mongolian cattle and Luxi cattle. **M** Creatine kinase content in the blood of WT and MT Mongolian cattle and Luxi cattle. **N** Bicarbonate content in the blood of WT and MT Mongolian cattle and Luxi cattle Figure 2A-C presented the blood glucose and lipids metabolism indicators of WT and MT Mongolian cattle at different pregnancy stages. Compared with the fertilization stage, the contents of Glu (3.34 ± 0.41 vs. 2.64 ± 0.63, *P* = 0.049) and LA (2.05 ± 0.45 vs. 1.73 ± 0.32, *P* = 0.324) in the blood of WT and MT Mongolian cattle increased and reached their peaks in the early gestation period. As pregnancy progressed, their contents gradually decreased, which was the same as that of Luxi cattle. Except for the third trimester, the Glu content in the blood of WT Mongolian cattle was higher than that of MT Mongolian cattle. During the first and third trimesters, the LA (2.74 ± 0.95 vs. 3.20 ± 0.33, *P* = 0.598, 1.84 ± 0.60 vs. 2.20 ± 0.29, *P* = 0.274) content in the blood of MT Mongolian cattle was higher than that of WT cattle, but there was no significant difference. No obvious change of LDH was observed in WT and MT Mongolian cattle at different pregnancy stages. Except for the first trimester (1199.67 ± 75.29 vs. 945.5 ± 9.5, *P* = 0.006), the blood LDH content of MT Mongolian cattle was higher than that of WT cattle (1146.44 ± 72.55 vs. 1268.67 ± 63.41, *P* = 0.039, 956 ± 77.65 vs. 1051 ± 66.35, *P* = 0.153, 816.5 ± 107.29 vs. 908.67 ± 136.45, *P* = 0.361), and there was a significant difference at fertilization stage. Compared with Luxi cattle, the blood LDH content of WT Mongolian cattle was higher than that of WT Luxi cattle at the fertilization stage and during the first trimester. Besides the first trimester, the blood LDH content of MT Mongolian cattle was higher than that of MT Luxi cattle. The results of lipid metabolism indicators were shown in Fig. 2D-G. During gestation, the blood TG (0.16 ± 0.02 vs. 0.31 ± 0.09, *P* = 0.001, 0.28 ± 0.07 vs. 0.27 ± 0.05, *P* = 0.485, 0.26 ± 0.06 vs. 0.37 ± 0.06, *P* = 0.03) content of WT and MT Mongolian cattle gradually increased. Except in third trimester (0.42 ± 0.06 vs. 0.36 ± 0.08, *P* = 0.301), the TG content in MT Mongolian cattle was higher than that of WT cattle. The contents of CHO (2.48 ± 0.32 vs. 2.03 ± 0.03, *P* = 0.001, 2.32 ± 0.05 vs. 2.82 ± 0.36, *P* = 0.009, 2.28 ± 0.05 vs. 2.28 ± 0.01, *P* = 0.938), HDL (2.69 ± 0.59 vs. 1.78 ± 0.09, *P* = 0.071, 3.39 ± 0.49 vs. 2.53 ± 0.033, *P* = 0.025, 2.74 ± 0.30 vs. 2.69 ± 0.46, *P* = 0.879) and LDL (1.03 ± 0.33 vs. 0.82 ± 0.12, *P* = 0.153, 1.25 ± 0.32 vs. 1.11 ± 0.18, *P* = 0.467, 1.10 ± 0.29 vs. 1.09 ± 0.08, *P* = 0.658) in the blood of WT and MT Mongolian cattle showed a trend of increasing first and then decreasing with the progress of pregnancy, but were all lower than the levels at the fertilization stage. Moreover, the contents of HDL and LDL in MT Mongolian cattle were lower than those of WT cattle at different pregnancy stages. The CHO content of MT Mongolian cattle was higher than that of WT cattle at fertilization stage and during the second trimester (2.23 ± 0.05 vs. 2.82 ± 0.36, *P* = 0.009). Compared with Luxi cattle, the blood TG content of WT Mongolian cattle was lower than that of WT Luxi cattle except in third trimester. During second and third trimester, the TG content of MT Mongolian cattle was higher than that of MT Luxi cattle. During the fertilization stage and the first trimester, the CHO content of WT Mongolian cattle was higher than that of WT Luxi cattle; while in the second and third trimester, the CHO content of MT Luxi cattle was lower than that of WT Luxi cattle. The blood HDL and LDL contents of WT and MT Mongolian cattle were lower than those of Luxi cattle at different pregnancy stages. The detection results of protein metabolism indicators were shown in Fig. 2H-J. At different pregnancy stages, the urea nitrogen (BUN) content in the blood of WT and MT Mongolian cattle did not show a significant trend of change. During fertilization (0.69 ± 0.24 vs. 1.45 ± 0.54, *P* = 0.008) and the first trimester (3.22 ± 0.74 vs. 3.66 ± 0.09, *P* = 0.46), the BUN content of MT Mongolian cattle was higher than that of WT Mongolian cattle, but it was lower in the second and third trimester. Except for fertilization period, the BUN content of WT Mongolian cattle was higher than that of WT Luxi cattle during gestation period. The blood BUN content of MT Mongolian cattle was lower than that of MT Luxi cattle, but this trend was not observed in the first and third trimesters. Compared with fertilization period, the blood TP and ALB contents in MT Mongolian cattle decreased at the early pregnancy stage, then increased and reached the highest level in the third trimester. The WT Mongolian cattle showed the same trend as Luxi cattle. With the extension of pregnancy cycle, the blood TP and ALB contents of Mongolian cattle increased first and then decreased. The results of the detection of blood health-related indicators in WT and MT Mongolian cattle were shown in Fig. 2K-N. Except for fertilization period, the blood AST content of MT Mongolian cattle increased first and then decreased as the pregnancy progressed, and the AST content of MT Mongolian cattle was lower than that of WT Mongolian cattle (54.93 ± 8.35 vs. 38.57 ± 5.24, *P* = 0.028, 49 ± 1.67 vs. 47.6 ± 4.68,*P* = 0.617, 57.53 ± 3.88 vs. 48.65 ± 0.75, *P* = 0.031). During fertilization period and the third trimester, the blood AST content of WT and MT Mongolian cattle was higher than that of Luxi cattle, while the opposite trend was observed during the first and second trimesters. The results showed that the inositol content in WT and MT Mongolian cattle gradually decreased as the pregnancy progressed, reaching the lowest level in the third trimester (133.78 ± 13.21 vs. 128.75 ± 7.19, *P* = 0.602). The blood inositol content of MT Mongolian cattle was lower than that of WT Mongolian cattle, and both were lower than that of Luxi cattle. Except the first trimester, the blood CK content of MT Mongolian cattle was higher than that of WT cattle at different pregnancy stages (133 ± 16.89 vs. 163 ± 29.16, *P* = 0.067, 122.83 ± 19.01 vs. 156.33 ± 29.49, *P* = 0.113, 80 ± 14.01 vs. 81.33 ± 29.89, *P* = 0.943). There was no significant trend change in the BC content of WT and MT Mongolian cattle. However, during first trimester, the blood BC content of MT Mongolian cattle was significantly lower than that of WT Mongolian cattle (22.25 ± 2.21 vs. 16.23 ± 1.05, *P* = 0.000). Moreover, as the pregnancy progressed, the BC content of MT Mongolian cattle increased, contrary to the situation of MT Luxi cattle. ### Serum reproductive hormone levels of WT and MT Mongolian cattle at different pregnancy stages To further explore the effect of MSTN gene editing on the pregnancy status of Mongolian cattle, we detected the contents of E2 and P4 in serum at different pregnancy stages. The results showed that the content of E2 in WT and MT Mongolian cattle decreased first and then increased throughout the pregnancy cycle. The E2 concentration was the lowest in the first trimester and then increased as pregnancy progressed. Moreover, the E2 content in serum of WT Mongolian cattle was higher than that of MT cattle throughout the pregnancy cycle (65.08 ± 2.56 vs. 59.12 ± 2.34, *P* = 0.129, 55.54 ± 1.80 vs. 51.84 ± 2.74, *P* = 0.186, 57.95 ± 1.37 vs. 56.12 ± 3.14, *P* = 0.491, 71.64 ± 2.56 vs. 66.11 ± 1.99, *P* = 0.126). Compared with WT and MT Luxi cattle, the blood E2 content of WT and MT Mongolian cattle was higher at different pregnancy stages (Fig. 3A). Unlike the changes in E2 levels during pregnancy, the P4 levels of WT and MT Mongolian cattle increased first and then decreased as gestation period extended. The P4 content was the highest in the first trimester (19.33 ± 0.54 vs. 20.74 ± 0.20, *P* = 0.050), followed by a continuous downward trend, and continued to decline in the second (21.12 ± 0.37 vs. 21.26 ± 0.49, *P* = 0.840) and third stages. Additionally, the P4 content in serum of MT Mongolian cattle at different pregnancy stages was higher than that of WT cattle. The change of blood P4 content in Mongolian cattle was consistent with that of Luxi cattle. However, the serum P4 contents of WT and MT Mongolian cattle were significantly lower than that of WT and MT Luxi cattle (Fig. 3B). Fig. 3Serum reproductive hormone levels of WT and MT Mongolian cattle and Luxi cattle at different pregnancy stages. **A** E2 levels in the serum of WT and MT Mongolian cattle and Luxi cattle. **B** P4 levels in the serum of WT and MT Mongolian cattle and Luxi cattle ### Analysis of blood metabolomics in WT and MT Mongolian cattle at different gestation stages Based on the above results, we found that there were differences between MT and WT Mongolian cattle in reproductive traits, blood physiological and biochemical indicators, and the content of reproductive hormones in serum. By detecting the blood metabolites of WT and MT Mongolian cattle at different pregnancy stages and comparing them with those of Luxi cattle, we further explored the impact of *MSTN* gene editing on the reproductive traits of Mongolian cattle. The results showed that the metabolites between MT and WT Mongolian cattle were clearly separated and significant difference existed. The OPLS-DA permutation validation of the blood metabolome of WT and MT Mongolian cattle at different pregnancy stages showed that as the permutation retention decreased, the R2 and Q2 values also decreased, and the regression line showed an upward trend. This indicated that the permutation validation was successful and the model did not exhibit overfitting (Figs. 4A and B, 5A and B, 6A and B and 7A and B). All of the above results indicated that there were significant differences in the blood metabolites between WT and MT Mongolian cattle at different pregnancy stages.Fig. 4Blood metabolomics analysis of WT and MT Mongolian cattle during the fertilization period. **A** OPLS-DA analysis of the blood metabolomics of WT and MT Mongolian cattle. **B** PLS-DA permutation validation of the blood metabolomics of WT and MT Mongolian cattle. **C** Cluster analysis of the top 20 differential metabolites in the blood metabolome of WT and MT Mongolian cattle. **D** Clustering analysis of differential metabolites in the blood of WT and MT Mongolian cattle during the fertilization period. **E **KEGG pathway enrichment analysis of the differential metabolites in the blood of WT and MT Mongolian cattleFig. 5Blood metabolomics analysis of WT and MT Mongolian cattle in the early pregnancy. **A** OPLS-DA analysis of the blood metabolomics of WT and MT Mongolian cattle. **B** PLS-DA permutation validation of the blood metabolomics of WT and MT Mongolian cattle. **C** Cluster analysis of the top 20 differential metabolites in the blood metabolome of WT and MT Mongolian cattle. **D** Clustering analysis of differential metabolites in the blood of WT and MT Mongolian cattle during the early pregnancy. **E **KEGG pathway enrichment analysis of the differential metabolites in the blood of WT and MT Mongolian cattleFig. 6Blood metabolomics analysis of WT and MT Mongolian cattle in the second trimester of pregnancy. **A** OPLS-DA analysis of the blood metabolomics of WT and MT Mongolian cattle. **B** PLS-DA permutation validation of the blood metabolomics of WT and MT Mongolian cattle. **C **Cluster analysis of the top 20 differential metabolites in the blood metabolome of WT and MT Mongolian cattle. **D** Clustering analysis of differential metabolites in the blood of WT and MT Mongolian cattle during the second of pregnancy. **E **KEGG pathway enrichment analysis of the differential metabolites in the blood of WT and MT Mongolian cattleFig. 7Blood metabolomics analysis of WT and MT Mongolian cattle in the third trimester of pregnancy. **A** OPLS-DA analysis of the blood metabolomics of WT and MT Mongolian cattle. **B** PLS-DA permutation validation of the blood metabolomics of WT and MT Mongolian cattle. **C** Cluster analysis of the top 20 differential metabolites in the blood metabolome of WT and MT Mongolian cattle. **D** Cluster analysis of differential metabolites in the blood of WT and MT Mongolian cattle during the trimester of pregnancy. **E** KEGG pathway enrichment analysis of the differential metabolites in the blood of WT and MT Mongolian cattle Under the conditions where the radioactive value is greater than or equal to 2 or less than or equal to 2, and when the VIP is greater than 1.0 and P is less than 0.05, the differential metabolites of WT and MT Mongolian cattle at different gestational stages were screened, and clustered was conducted (Figs. 4C, 5C, 6C and 7C). The cluster analysis of the top 20 differentially expressed metabolites in the blood metabolome during the fertilization period showed that, compared with WT Mongolian cattle, only 3 metabolites were up-regulated in MT Mongolian cattle, namely Perfluorohexane sulfonic acid, Momilactone B, and (1 S, 2 S, 4 S, 5R)−1,8-Epoxy-p-menthane-2,5-diol. The down-regulated differential metabolites mainly include glycine, aspartate-serine, ethyl β-D-glucuronide, L-kynurenine, 5-hydroxyindoleacetic acid, etc. (Fig. 4D). The KEGG pathway enrichment analysis of the enriched differential metabolites indicated that the differential metabolites of WT and MT Mongolian cattle during non-pregnant period were mainly enriched in signaling pathways, such as ABC transporters, purine metabolism, phenylalanine metabolism, tryptophan metabolism, glycerophosphollipid metabolism, 2-oxocarboxylic acid metabolism, and biosynthesis of amino acids (Fig. 4E). The metabolic differences between MT and WT Mongolian cattle during the first trimester were screened and enriched analyzed. The results showed that compared with WT Mongolian cattle, the down-regulated metabolites in MT Mongolian cattle included Aspartyl-Serine, Pimelic acid, Glycodeoxycholic acidy, and 3a,7b,12a-Trihydroxyoxocholanyl-Glycine, while the up-regulated differential metabolites included Perfluorohexane sulfonic acid and Pyroglutamine (Fig. 5D). The KEGG pathway enrichment analysis indicated that the differentially expressed metabolites in the blood metabolome of WT and MT Mongolian cattle during the early pregnancy were mainly enriched in signaling pathways such as sphingolipid signaling pathway, phenylalanine metabolism, glycerophospholipid metabolism, arachidonic acid metabolism, and linoleic acid metabolism (Fig. 5E). The screening and clustering analysis of the differential blood metabolites of WT and MT Mongolian cattle in the second trimester revealed that, compared with WT Mongolian cattle, the levels of D-(-)-mannitol, m-chlorobenzoic acid, and 3-methyldioxyindole were elevated in MT Mongolian cattle, while the metabolites such as DL-indole-3-lactic acid, ethyl.β-D-glucuronide, 4-methylcatechol, (3 − phenylpropionyl)glycine, and 3-hydroxy-4-aminopyridine sulfate were downregulated in MT Mongolian cattle (Fig. 6D). The KEGG pathway enrichment analysis showed that these differential metabolites were mainly enriched in pathways such as metabolic phenylalanine metabolism, tryptophan metabolism, glycerolipid metabolism and glycerophospholipid metabolism (Fig. 6E). During the third trimester of pregnancy, the levels of metabolites such as aniline, thymine, L-kynurenine, 5-hydroxyindoleacetic acid, aspartyl-leucine and uric acid were downregulated in MT Mongolian cattle, while the levels of metabolites such as deoxycholic acid, ursocholic acid and cholic acid were upregulated (Fig. 7D). The KEGG pathway enrichment analysis showed that these differential metabolites were mainly enriched in signaling pathways such as citrate cycle (TCA cycle), purine metabolism, lysine degradation, phenylalanine metabolism, tryptophan metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, 2-oxocarboxylic acid metabolism, and biosynthesis of amino acids (Fig. 7E). The above results of blood metabolomics of WT and MT Mongolian cattle at different pregnancy stages indicated that, compared with WT Mongolian cattle, the differential blood metabolites of MT Mongolian cattle were mainly related to fatty acid synthesis, the synthesis and metabolism of various amino acids, and the TCA cycle. This is consistent with the previous findings that there are abnormalities in amino acid metabolism and protein synthesis in the blood metabolome of MT Luxi cattle. These results suggest that *MSTN* gene-edited Mongolian cattle may have relatively better protein synthesis and metabolism capabilities. ## Discussion Gene editing technology is a powerful tool that is widely used in the field of biological breeding. Through the application of gene editing technology, significant progress has been made in the breeding of superior traits in domestic animals such as goats, sheep, pigs and cattle [19–21]. *MSTN* gene editing can be used to increase meat production and lean meat percentage of beef cattle. Under the same feeding conditions, after *MSTN* gene editing, the meat production rate can be increased by 1.3 times. The fat content in the lean meat is significantly reduced, while the content of polyunsaturated fats increase [22]. In cattle, the mutation of *MSTN* gene not only brings about excellent meat production, but also has a certain impact on the reproductive capacity of cattle. The bone weight of *MSTN* gene-edited cattle has decreased. In particular, hip joint dysplasia may have an impact on reproductive performance [23]. Wiener et al. found in their study that the incidence of dystocia in Charolais cows carrying the *MSTN* gene mutation increased, and the mortality rate of calves was also higher. This might be due to the reduction in pelvic area, prolonged gestation period, and increased birth weight of calves [24]. In Belgian Blue cattle and Aquitaine cattle, the birth weight of calves carrying *MSTN* gene mutations increased significantly [25, 26]. In this study, it was found that the dystocia rate of MT Mongolian cattle was significantly lower than that of WT Mongolian cattle, and the calf survival rate of MT Mongolian cattle was slightly higher than that of WT Mongolian cattle. The study found that the pelvic area of MT Mongolian cattle is relatively smaller, but the birth weight of their calves is significantly higher than that of WT Mongolian cattle. However, the dystocia rate of MT Mongolian cattle is lower. This may be because the editing of the MSTN gene affects the uterine contraction ability of the cows. Meanwhile, it may also change the body proportion and flexibility of the fetuses. The combined action of multiple factors reduces the dystocia rate of MT Mongolian cattle. In addition, the size and condition of reproductive organs such as the ovaries and uterus are also the main factors affecting the reproductive performance of cattle. Cows with larger ovaries have a greater reproductive advantage because larger ovaries can accommodate more dominant follicles. The follicular fluid has a relatively high content of energy substances and lipids, which is more conducive to successful pregnancy after breeding [27]. The reduction in the number of ovaries was observed in *MSTN* gene-edited cattle [28] and mice [29]. Further research revealed that the quality of oocytes and the developmental capacity of the embryos were not affected. In this study, the ovarian size and uterine diameter of WT and MT Mongolian cattle were measured. The results showed that there was no significant difference in ovarian size between WT and MT Mongolian cattle. The right ovary of MT Mongolian cattle was larger, but its size was smaller than that of Luxi cattle, which may be related to the relatively small body size of Mongolian cattle. An increase in uterine diameter has an adverse effect on the reproductive capacity of cows. The smaller the uterine diameter, the more thorough the remodeling progress [30]. This study found that the uterine diameter of MT Mongolian cattle was slightly larger than that of WT Mongolian cattle, but the difference was not significant. The above results indicate that the edition of the *MSTN* gene has no effect on the reproductive organs and reproductive indicators of Mongolian cattle. Moreover, compared with Luxi cattle, Mongolian cattle have better reproductive performance. Basarab et al. reported that when the ratio of the pelvic area of cows to the birth weight of calves exceeds 4.5, the dystocia rate decreases, and a positive correlation exist [31]. The detection in this study found that the ratios of pelvic area to birth weight for both WT and MT Mongolian cattle were higher than 4.5. However, the ratio of pelvic area to birth weight for MT Mongolian cattle was significantly lower than that of WT Mongolian cattle, which might be the reason for the lower dystocia rate in the MT Mongolian cattle. Previous studies mainly focused on the changes in metabolites in the muscles of *MSTN* gene-edited cattle. Mutation of *MSTN* gene can enhance the glucose metabolism level in muscle tissue by promoting the processes of glycolysis and the tricarboxylic acid cycle [32]. In this study, we observed that the levels of Glu and LA in the blood of MT Mongolian cows showed a trend of first increasing and then decreasing during pregnancy. This might be due to the changes in the LDH content in the blood at different stages of pregnancy. Moreover, the analysis of the blood metabolome during the third trimester showed that the differential metabolites were related to the TCA cycle. The levels of serum Glu and LA of MT Mongolian cows in the first trimester were relatively high. This phenomenon may be due to the larger body weight of the *MSTN*-edited fetuses, which require more energy consumption. Larger fetuses require more energy to support their subsequent development and to maintain the cow’s parturition. Therefore, the cows will correspondingly produce more energy-related substances, which may also be one of the reasons for the lower dystocia rate in MT Mongolian cows. This further reminds us that more attention should be paid to the energy supply of *MSTN* cows during pregnancy to ensure the normal delivery of the fetuses. The *MSTN* gene mutation has a dual impact on beef cattle production. On one hand, the *MSTN* gene mutation significantly increases the carcass weight by boosting muscle production. On the other hand, the *MSTN* mutation also leads to a decrease in the intramuscular fat content of the carcass [33]. In this study, the lipid-related indicators of both MT and WT Mongolian cows showed a trend of first increasing and then decreasing. The accumulation of lipids in the first trimester provides a large amount of energy for the maintenance of pregnancy, thus avoiding negative energy balance during pregnancy. It also plays important roles in normal development of the fetus, the smooth parturition of cows, the healthy growth of calves and postpartum recovery. Total protein and albumin are indicators reflecting the synthetic ability of the liver. The total protein and albumin in the blood of MT Mongolian cows were significantly lower than those of WT Mongolian cows during the first and second trimester, and this phenomenon was restored in the third trimester. This might be due to the fact that in the early pregnancy, MT Mongolian cows need to consume a large amount of energy to promote the development of the fetus. The changes in the levels of E2 and P4 in the blood of cows during pregnancy play a crucial role in maintaining pregnancy. E2 is mainly synthesized and secreted by ovaries, corpora lutea, and the placenta during pregnancy. Before pregnancy, it partipates in the regulation of cow’s estrus. An increase in E2 concentration promotes the secretion of luteinizing hormone (LH). Subsequently, LH and FSH act synergistically to further promote the growth and maturation of follicles, thereby ensuring the success of pregnancy [16]. Previous studies have shown that the expression of the *MSTN* gene promote the secretion of E2 and inhibit the production of P4 in the ovaries, thereby reducing the estrus rate of cows [34]. In this study, detection of the blood E2 content of WT and MT Mongolian cows at different pregnancy stage indicated that *MSTN* gene editing reduced the E2 content. The E2 content in both WT and MT Mongolian cows showed a trend of first decreasing and then increasing throughout the pregnancy cycle. The E2 concentration was the lowest in the first trimester and then increased as pregnancy progressed. Moreover, the E2 content in the serum of WT Mongolian cows was higher than that of MT cows throughout the pregnancy cycle. High-level E2 in the third trimester may play a key role in maintaining the normal function of the cow’s reproductive system, promoting the parturition process, and assisting postpartum recovery. Throughout the entire pregnancy period, the blood P4 content of MT Mongolian cows was higher than that of WT Mongolian cows, but with no significant difference. Both showed a gradually increasing trend as the pregnancy progressed, which provided necessary support for embryo implantation and fetus development. The *MSTN* gene can stimulate protein degradation through the proteasome ubiquitin activation mediated by Forkhead box family proteins 1 and 3 (FoxO1/3). After the *MSTN* gene is edited, more proteins will be involved in muscle synthesis and protein degradation will decrease [35]. This study also found that the total protein content in the blood of MT Mongolian cows was higher than that in WT Mongolian cows during pregnancy. Moreover, analysis of the blood metabolome at different pregnancy stages showed that the differential metabolites were mainly enriched in amino acid metabolic pathways, which further confirmed that *MSTN* gene editing reduced protein degradation. Tryptophan is an essential amino acid for ruminants. In cattle, after a portion of tryptophan is degraded by rumen microorganisms, there are mainly four utilization pathways, namely protein synthesis, the serotonin pathway, the kynurenine pathway, and degradation by intestinal microorganisms [36]. By analyzing the blood metabolome of WT and MT Mongolian cows at different pregnancy stages, we found that significant differences in downstream metabolites of tryptophan such as kynurenine and indole compounds, between MT and WT Mongolian cows. This might also be the reason for the abnormal protein synthesis in the MT Mongolian cows. The substrates for nucleotide catabolism include purines and pyrimidines. Pyrimidine nucleotides synthesized from pyrimidines and ribose can be phosphorylated to produce uridine triphosphate (UTP). UTP acts in concert with glucose-1-phosphate to generate uridine diphosphate glucose (UDP-glucose), which participates in glycogen synthesis. During the third trimester, the rapid development of the fetus requires a large amount of energy provided by the cow through fat mobilization and nucleotide metabolism [37]. This study found that nucleotide metabolism decreased in MT Mongolian cows during the third trimester. This might be because MSTN gene editing promoted lipid metabolism, providing more substrates for glycolysis. Further analysis of the metabolomics results revealed that the concentrations of various organic substances in the blood of Mongolian cows were higher during the third trimester. This proves that Mongolian cows can provide sufficient energy for parturition, lactation, and fetal development, ensuring the health of the cow and fetus as well as postpartum recovery. This might also be one of the reasons why the dystocia rate of Mongolian cows is lower than that of Luxi cows. In summary, through a comprehensive analysis of the reproductive phenotypes, blood physiological and biochemical indicators, reproductive hormones, and blood metabolome of WT and MT Mongolian cattle, and by comparing them with Luxi cattle, the feasibility of applying *MSTN* gene editing in the breeding of different breeds of beef cattle was laid a foundation. ## Conclusions This study indicates that *MSTN* gene editing has no significant effect on the estrus rate, pregnancy rate, calf birth rate, and survival rate of Mongolian cattle, and also does not change the sizes of ovaries and uterus. Moreover, the dystocia rate of MT Mongolian cattle is significantly lower than that of WT Mongolian cattle. Analyses of blood physiological and biochemical indicators, reproductive hormone levels and blood metabolomics indicate that, compared with WT cattle, the *MSTN*-edited Mongolian cattle has increased glucose metabolisms, decreased lipid synthesis, improved protein synthesis, reduced tryptophan metabolism, and decreased E2 and increased P4 concentration. Moreover, Mongolian cattle exhibit better reproductive performance than Luxi cattle, which can be considered as potential breeds for subsequent beef cattle breeding. ## Materials and methods ### Animals The WT and MT Mongolian cattle selected in this study were all from the Grassland Livestock Germplasm Innovation and Breeding Base of Inner Mongolia University in Helinge’er New Area, Hohhot. The WT Mongolian cattle were artificially inseminated Mongolian cattle with synchronized estrus. The construction process of MT Mongolian cattle can be found in the study of Zhao et al. [38]. In brief, using the CRISPR-Cas9 gene-editing technology, the *MSTN* gene was targeted, and specific knock-outs were carried out at different sites of the *MSTN* gene. Subsequently, Mongolian cattle with the *MSTN* gene knocked out were successfully bred through somatic cell cloning technology. All cattle were raised under the standard feeding conditions of the ranch. The feed components and nutritional ingredients are shown in Additional file 1 Table S1. The reproductive data of 100 WT and 100 MT Mongolian cattle were statistically analyzed respectively. The selected Mongolian cattle were all between 2 and 2.5 years old, had never given birth, were in good health and physiological condition, and were capable of normal estrus. Their reproductive data were collected and analyzed. The gestation period classification can refer to the report by Zhao et al. [17]. The gestation period of the cows is generally 285 days. The day of artificial insemination was recorded as Day 0 of pregnancy. The period before insemination was considered the non-pregnant period, 0–95 days was recorded as the first trimester, 95–190 days was the second trimester, and 190 days until the day of calving was the third trimester. Additionally, 10 WT and 10 MT Mongolian cows were selected from each group to measure their reproductive organs and collected blood for physiological and biochemical analysis. Before insemination, blood was collected from the tail vein of the cows, and the serum was separated by centrifugation. Meanwhile, an ultrasound device was used to measure the sizes of the ovaries, pelvis, and uterus. On Day 33, PGF was administered intramuscularly. Estrus status of the cows was observed and recorded 24 h later. Artificial insemination was carried out 12 h after with two consecutive inseminations at an interval of 8–10 h. Pregnancy status was checked and blood was collected 60 days after insemination. Subsequently, blood was collected from pregnant cows on Days 150 and 250. Physiological and biochemical tests were performed on the blood samples collected at different pregnancy stages. After calving, the birth rate of calves and the dystocia rate of cows were statistically analyzed. Finally, 6 WT and 6 MT Mongolian cows were selected respectively to conduct blood metabolomics analysis during the insemination period, early pregnancy, middle pregnancy, and late pregnancy periods. ### Measurement of pelvic sizes The method for measuring the size of pelvis can be found in Zhao et al. (*n* = 10) [17]. In brief, before measuring the pelvis, the external genitalia and anal area of the cow need to be thoroughly cleaned to avoid contamination. Use customized pelvic caliper to measure the height and width of the pelvic cavity. The maximum distance between the two pelvic bones is the width of the pelvic inlet; the distance from the ischial tuberosity at the bottom of the pelvis to the nearest point at the bottom of the sacrum is the height of the pelvic inlet; the product of the width and height of the pelvic inlet is the area of the pelvis. During the measurement process, the two ends of the caliper should not leave the measurement point, and gentle force should be applied to avoid damaging the uterus. ### Measurement of ovarian size and uterine diameter The ovarian sizes of WT and MT Mongolian cattle (*n* = 10) were measured using a B-ultrasound instrument. The method for measuring the uterine diameter was based on the description provided by Baze et al. [39]. Specifically, a portable ultrasound device (Ibex pro; E.I. Medical Imaging, Loveland, CO, USA) equipped with a 7.5 MHz linear array probe was used to measure the uterine diameter. The uterine diameter was defined as twice the distance from the center point of the uterine horn to the boundary of the endometrial echo, and was measured on two different vertical cross-sections at each horn. ### Measurement of blood physiological and biochemical indicators in WT and MT Mongolian cattle at different pregnancy stages Blood samples were collected from the tail veins of WT and MT Mongolian cattle in a fasting state at different pregnancy stages. The blood samples were centrifuged at 3500 rpm for 15 min. The upper-layer serum was separated and collected, and then stored at −80 °C. The physiological and biochemical parameters of the blood were detected using the MSCAN-Ⅱdry analyzer. The physiological and biochemical parameters included Glu, LA, LDH, TG, CHO, HDL, LDL, BUN, TP, ALB, AST, CK and BC. ### Detection of serum E2 and P4 concentrations in WT and MT Mongolian cattle at different pregnancy stages The blood hormone levels of three WT Mongolian cattle and three MT Mongolian cattle at different stages of pregnancy were analyzed respectively. According to the manufacturer’s instruction, the bovine progesterone ELISA kit (MM-5091802, MEIMIAN, Jiangsu, China) and the bovine estradiol ELISA kit (MM-002302, MEIMIAN, Jiangsu, China) were used to detect the levels of E2 and P4 in the serum of WT and MT Mongolian cattle at different pregnancy stages. Briefly, these columns were equilibrated at room temperature for 20 min. Standard and sample wells were set up respectively. Fifty microliters of different concentrations of standard solution were added to each standard well. For the sample wells, 10 µL of the sample was added first, followed by 40 µL of sample diluent. Additionally, a blank well was set up. Except for the blank well, 100 µL of the antibody labeled with horseradish peroxidase (HRP) was added to each of the standard wells and the sample wells, and incubated at 37 °C for 60 min. The liquid was discarded, and each well was added with washing solution and left to stand for 1 min. Then the washing solution was discarded. The plate washing steps were repeated 5 times. Added 50 µL of Substrate A and 50 µL of Substrate B to each well respectively, and incubated at 37 °C in the dark for 15 min. Then, 50 µL of the stop solution was added to each well to terminate the reaction. The optical density (OD) values of each well were measured using a microplate reader (Thermo, USA) at a wavelength of 450 nm within 15 min. ### Analysis of blood metabolomics of WT and MT Mongolian cattle at different pregnancy stages Six WT Mongolian cattle and six MT Mongolian cattle were selected respectively. Blood was collected from these cattle and serum was separated for blood metabolomics detection and analysis. Twenty microliters of serum samples of WT and MT Mongolian cattle were dropped respectively to different wells, and then 120 µL of 50% methanol (A-456-4, Fisher, USA) was added for thorough mixing. The samples were then left at room temperature for 10 min to extract metabolites. The extracts were placed at −20 °C overnight to precipitate the proteins. The samples were centrifuged at 4000 g for 20 min, and the supernatant was transferred to a 96-well plate. Taken 10 µL from each sample and mixed with the diluent to form quality control (QC) samples. The extracted samples were sequenced on a machine. The raw data was converted to readable mzXML data using Proteowizard and MSConvert software, and peak extraction and quality control were performed using XCMS software. The adduct ions of the extracted substances were annotated by CAMERA software, and the data was preliminarily identified by metaX (v2.0) software. The identification information was matched with the in-house standard database. The enriched metabolites were annotated through databases such as HMDB and KEGG. Differentially expressed metabolites were screened based on univariate statistical analysis (t-test), combined with multivariate statistical analysis (OPLS-DA/PLS-DA) and fold change value (FC). The default screening conditions were *P* < 0.05, VIP > 1, and (FC < 1 or FC > 1). ### Data analysis The original data of estrus rate, pregnancy rate, calf birth rate and survival rate of WT and MT Mongolian cattle, as well as the ovarian and uterine sizes and pelvic area, were all presented in the form of mean ± standard deviation. The data were analyzed for significance using SPSS 22.0 software (one-way ANOVA was used to compare multiple groups, and Student’s t-test was used to compare between two groups). A *P*-value < 0.05 was considered to indicate a significant difference. In the charts, (*) indicates a significant difference between the two groups, where **P* < 0.05, ***P* < 0.01, ****P* < 0.001, while “ns” indicates no significant difference. In the tables, different letters indicate significant difference between the two groups (*P* < 0.05), and the same letters indicate no significant difference (*P* > 0.05). The charts were drawn using GraphPad Prism 8 software. ## Supplementary Information Additional file Table S1 Feed Ingredients and Nutritional Composition. Additional file 2.