Semen quality and fertilization ability of myostatin-knockout boars

Semen quality and fertilization ability of myostatin-knockout boars

Theriogenology 135 (2019) 109e114 Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com Semen quality an...

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Theriogenology 135 (2019) 109e114

Contents lists available at ScienceDirect

Theriogenology journal homepage: www.theriojournal.com

Semen quality and fertilization ability of myostatin-knockout boars Sheng-Zhong Han 1, Song-Shan Jin 1, Mei-Fu Xuan, Qing Guo, Zhao-Bo Luo, Jun-Xia Wang, Jin-Dan Kang**, Xi-Jun Yin* Jilin Provincial Key Laboratory of Transgenic Animal and Embryo Engineering, Yanbian University, Yanji, Jilin, 133002, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 March 2019 Received in revised form 16 May 2019 Accepted 30 May 2019 Available online 31 May 2019

There is no data currently available on the semen quality and fertility of myostatin-knockout (MSTN/) boars. We showed that sexually mature adult homozygous MSTN mutant boars have an obvious “double muscling” phenotype, along with a MSTN/ boar head, back, abdomen, eyes, and oral cavity. Additionally, no abnormalities were found in the reproductive organs. The semen color, odor, and pH also had no abnormalities. The MSTN/ boars showed good sexual desire. The concentration, motility, plasma membrane integrity, deformity, acrosome integrity, and mitochondrial activity of the semen presented no significant differences from those of the control semen (Duroc). The ejaculation volume of the MSTN/  boars was significantly lower than that of the control (168.78 ± 6.70 and 223.11 ± 21.21 mL, respectively). The rate of cleavage and blastocyst between the MSTN/ and control boar semen were compared by in vitro fertilization. The results showed that in the eggs fertilized by the MSTN/ boar semen, the two-cell and blastocyst rates were similar to those of the control semen (69.1 ± 0.7% vs 65.2 ± 1.6% and 20.2 ± 1.2% vs 22.8 ± 1.4% for the two-cell and blastocyst rates, respectively). In this study, nine healthy offspring were successfully produced through artificial insemination using the MSTN/ boar semen. Thus, an MSTN/ boar can be used as the terminal male parent and is expected to be developed into new super lean meat varieties in the future. © 2019 Elsevier Inc. All rights reserved.

Keywords: Myostatin-knockout Boar Semen quality In vitro fertilization

1. Introduction Myostatin (MSTN), also known as the growth and differentiation factor 8 (GDF8), regulates the homeosstasis of the skeletal muscle tissue by inhibiting skeletal muscle growth [1,2]. The natural gene mutation occurred in some cattle breeds, for example, the Belgian Blue (BB) is a relatively recently established cattle breed, characterized by extreme conformation - commonly known as “double muscling”. Double muscling is due to a mutation of the MSTN gene. MSTN mutations are pleiotropic in their effects, meaning that they affect a number of different body systems. Some of these gene effects are known to negatively impact the welfare of cattle. These issues are so well known and accepted that even those who promote the increased lean meat production of the breed for beef farming advise producers not to set up herds of pure-bred BBs.

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (J.-D. Kang), [email protected] (X.-J. Yin). 1 Sheng-Zhong Han and Song-Shan Jin contributed equally to this study. https://doi.org/10.1016/j.theriogenology.2019.05.047 0093-691X/© 2019 Elsevier Inc. All rights reserved.

Instead, they recommend using the BB breed as a sire to cross with other non-double muscled breeds [3]. In programs where bulls were evaluated for their breeding soundness, 93.7% of young BB bulls failed compared to 59.3% of young Holstein Friesian (HF) bulls. This was mainly due to a poor semen morphology and small testicular circumference [4]. In a previous study, the semen quality of BB and HF bulls, in several age categories, was compared by means of a classical semen evaluation. Volume and concentration, and consequently the total semen output, depended largely on age. The gross, total, and progressive motility, along with the % live and % normal spermatozoa were significantly lower in the BB breed. Primary semen abnormalities, such as nuclear vacuoles, midpiece defects, and cytoplasmic droplets, which were noticed most frequently, occurred far more in the BB breed. Hence, disturbances in spermatogenesis are deemed to be the cause of the poorer BB semen quality. Since these semen abnormalities occur significantly more in the BB breed than in the HF breed, it seems that the BB breed is genetically predisposed to be more susceptible to environmental stresses, which are known to interfere with normal spermatogenesis [5]. Recently, our laboratory generated homozygous MSTN/ boars, using a new genome-editing technology in combination with the

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SCNT. These MSTN/ boars developed and grew normally to sexual maturity, showing ‘double muscle’ phenotypic characteristics as observed in the BB breed [6]. There is no data currently available on the semen quality and fertility of MSTN/ boars. The present study compared the semen quality of MSTN/ boars to wild type boars, by the physical characteristics of the semen, semen quality, and in vitro fertilization ability. Additionally, we tested the in vivo fertility after artificial insemination. 2. Materials and methods 2.1. Ethics statement All procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory animals. The animal experimental procedures of this study were approved by the committee on the Ethics of Animal Experiments at Yanbian University (Approval ID: 20130310). 2.2. Animals and management Boars were housed in temperature-controlled rooms and placed in a barn (2.1  2.3 m) with a slatted floor. Diets of 1.8 kg were fed on an individual basis, twice daily, and water was provided ad libitum. At weaning, sows were moved to an individual gestation stall (0.60  2.10 m), which had a concrete slatted floor, and were mated at weaning after estrus. The semen collected from MSTN/ boars was provided for artificial insemination (AI) of the sows. During the gestation period, diets of 1.4 kg were fed on an individual basis, twice daily. After 110 d of gestation, the pregnant sows were washed and moved into farrowing crates (2.4  1.8 m). After farrowing, the lactating diet was increased gradually until 5 d postpartum and then provided ad libitum. Weaning was done at approximately 24 d. 2.3. Physical measurements and reproductive examination Body measurements were taken using a measuring tape and modified caliper. Body measurement included the body length, body height, chest girth, chest width, abdominal girth, hip girth, hip width, hip length, and cannon circumference. The backfat thickness at the P2 position of the boar was measured. The general health, head, back, abdomen, eyes, and oral cavity were examined. The testicle size was measured using Vernier calipers and the epididymides and scrotum were examined. The penis and prepuce should be examined for abnormalities during the semen collection. 2.4. Semen collection and evaluation The ejaculate was collected at weekly intervals, by the same trained technician using the gloved-hand technique. The semen was collected into a pre-warmed plastic container (38  C), which was covered with a filter. There were nine ejaculates collected from the boar. Immediately after collection, the ejaculates were used to determine the following physical characteristics: the ejaculate volume (mL), ejaculate time (min), odor, color, and pH of the semen. The semen concentration and motility were counted by the CASA system (Tongfang, China). The collected semen was sent to the laboratory within half an hour, diluted, and immediately tested. The semen and slides were pre-warmed at 38  C, prior to CASA analysis. Analyses were performed on four microscope fields, with a total of at least 200 cells analyzed per sample. The semen motility variables measured were the total motility, velocity average line (VCL), velocity average path (VAP), velocity straight line (VSL), amplitude of the lateral head displacement (ALH), straightness

(STR), linearity (LIN) and wobble (WOB). Semen acrosomal integrity and deformity were measured using Coomassie blue and Eosin stained smears, respectively. First, fresh semen was applied evenly onto clean slides, then fixed with 4% paraformaldehyde, after natural air drying. Secondly, the slides were stained with either Coomassie blue or Eosin for 30 min, and then stained with dye that was washed away with a gentle water flow.The slides were observed under a 1000  light microscope (Olympus, IX71, Japan) after natural air drying. Between 200 and 300 spermatozoa were evaluated in each sample. Hoechst33342, PI, and JC-1 (5,50 ,6,60 -tetrachloro-1,10,3,30 -tetraethyl benzimidazolyl carbocyanine iodide; Invitrogen, Milan, Italy) were used for the simultaneous evaluation of the semen plasma membrane integrity, at the level of the semen head, and mitochondrial activity, over the midpiece. Hoechst 33342 and PI are DNA-specific dyes that stain all cells blue (Hoechst 33342) and dead cells red (PI). JC-1 has the unique ability to differentially label mitochondria with a high or low mitochondrial transmembrane potential (MMP). In mitochondria with a high MMP, JC-1 forms multimeric aggregates that emit bright orange, however, JC-1 forms monomers within mitochondria with a low MMP, emitting green. For analysis, 2.5 mL of 20 mmol/L Hoechst 33342 in dimethyl sulfoxide (DMSO) and 5 mL of 153 mmol/L JC-1 in DMSO were added to 500 mL of a prewarmed (37  C) semen sample and incubated at 37  C for 20 min, under lightproof conditions. Then, 2.5 mL of 2.4 mmol/L PI in water was added and the samples were incubated at 37  C for 10 min. The stained spermatozoa were observed as detailed by the above description using an epifluorescence microscope. Between 200 and 300 spermatozoa were evaluated in each sample. 2.5. In vitro fertilization For mTBM: 3 mM of KCl, 113.3 mM of NaCl, 5 mM of sodium pyruvate, 7.5 mM of CaCl2$2H2O, 11 mM of glucose, and 20 mM of Tris were dissolved in PBS. For the semen wash medium, 2 mg/ml of BSA and 150 mg/ml of streptomycin were dissolved in mTBM. For semen fertilization medium: 2 mM of caffeine and 4 mg/ml of BSA were dissolved in mTBM. For oocytes, cumulus cells were removed, denuded oocytes were washed three times in the IVF mTBM medium, and 30 oocytes were put into 50 mL of the IVF medium. For semen, diluted semen was washed in the semen wash medium and then washed in the semen fertilization medium two times for 4 min at 1200 g (final concentration is 1  105e1  106 in 50 mL of IVF mTBM medium). For IVF, 50 mL of semen, which had been washed in the semen fertilization medium, was put into 50 mL of the IVF medium that contained 30 oocytes, this was left to culture for 6e8 h at 38.5  C, with 5% CO2. Finally, fertilized embryos were cultured in the PZM medium in vitor for 2e6 d, to observe the development of cleavage and blastocyst. 2.6. Artificial insemination and reproductive performance Estrus detection was performed at 8:00 a.m. and 4:00 p.m. every day from 3 d after weaning using a boar. Sows (Landrace  Yorkshire) were considered to be in estrus when they showed a standing response. Sows were artificially inseminated two times, with a 12 h interval. 2.7. Statistical analysis Data are presented as the mean ± SEM and are derived from at least three independent experiments. Groups were compared using the Student's t tests, with P < 0.05 considered statistically significant.

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3. Results 3.1. General characteristics of the MSTN/ boars /

The MSTN boars were produced using the SCNT, using donor cells that had 2 and 4 bp deletions in the two alleles in the first exon of the myostatin gene [6]. This experiment was carried out by one MSTN/ boar, whose phenotype is characterized by hypertrophy of muscles, most prominently in the regions of the proximal fore and hind quarters, and prominent muscular protrusion, with intermuscular boundaries and grooves clearly visible beneath the skin (Fig. 1A). There is no abnormality in the back or belly (Fig. 1A). No abnormalities were detected in the testicles, epididymides, or scrotum (Fig. 1A). Additionally, the penis and prepuce are also normal (Fig. 1B and C). Although less froth and taint were observed in the MSTN/ boars, this was not related with the libido. Furthermore, no abnormalities were observed in the eyes or oral cavity. As the MSTN/ boars found it difficult to jump up to the dummy sow, semen was collected using the gloved hand technique by the same experienced technician after training the boar. The body length of the adult MSTN/ boar was 152 cm, body height was 88 cm, and heart girth was 151 cm. Hip girth, breadth, and length were 124, 45, and 43 cm, respectively. The cannon circumference was 21 cm. The backfat thickness was 12.4 mm. The testis width and length were 20.3 and 21.3 cm, respectively. 3.2. Assessment of the physical parameters of the semen of the MSTN/ and control (duroc) boars As shown in Table 1, the ejaculate volume of the MSTN/ boar was significantly lower than that of the control (168.78 ± 6.70 and 223.11 ± 21.21 mL, respectively). However, the total time, concentration, total semen, semen motility rate, and pH of the MSTN/ boars were not significantly different from those of the control (Table 1). The color of the semen of the MSTN/ boars was milkwhite (Fig. 1D) and the odor was normal, resembling a fishy smell. 3.3. Motility assessment, using the CASA system, for the MSTN/ and control boars /

The MSTN boar showed semen motility similar to that of the control. The VCL, VSL, VAP, ALH, LIN, WOB, and SRT of the MSTN/ boar semen were not significantly different from those of the

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Table 1 Evaluation of the physical characteristics of the semen of the MSTN/ and control boars (n ¼ 9). Item

WT

MSTN/

Ejaculation time (min) Ejaculation volume (mL) Concentration (108/mL) a Total semen (109) Motility (%) pH

7.53 ± 0.31 223.11 ± 21.21a 3.99 ± 0.40 868.17 ± 86.67 89.23 ± 2.14 7.18 ± 0.04

6.83 ± 0.23 168.78 ± 6.70b 4.53 ± 0.28 756.63 ± 42.66 91.03 ± 2.75 7.13 ± 0.05

a Total number of spermatozoa. Values are expressed as the mean ± SEM of nine replicates. Different letters in a row indicate a significant difference at the P < 0.05 level.

control (Table 2). 3.4. Assessment of the semen quality of the MSTN/ and control boars using staining The assessment of the morphology and acrosome status of the semen are shown in Fig. 2A and B. The assessment of the viability and mitochondrial activity of the semen are shown in Fig. 2C. Our results show that the viability, deformity rate, acrosome integrity, and high mitochondrial activity of the ejaculates were not significantly different between the MSTN/ and control boar semen (Table 3). 3.5. Comparison of the development of IVF embryos induced from the MSTN/ and control boar semen Our results showed that the cleavage rate of the MSTN/ boar was not significantly different compared to that of the control (65.22 ± 1.59 and 69.10 ± 0.69, respectively). The blastocyst of IVF was shown in Fig. 3, the blastocyst rate was not significantly different in the MSTN/ and control boars (22.83 ± 1.42 and 20.24 ± 1.21, respectively) (Table 4). 3.6. Artificial insemination of piglets from MSTN/ boar semen We artificially inseminated two sows, one of which gave birth to four piglets and the other to five (Fig. 4). The five F1 offpring had an average birth weight of 1.53 ± 0.02 kg, and a weaning weight of 6.15 ± 0.06 kg. 4. Discussion The double muscling (DM) phenotype is characterized by hypertrophy of the muscles, most prominently in the regions of the proximal fore and hind quarters, and prominent muscular protrusion, with intermuscular boundaries and grooves clearly visible beneath the skin [7]. These characteristics are present in MSTN/ boars. A general physical examination should be part of every

Table 2 Evaluation of the semen motility in the MSTN/ and control boars, using CASA system (n ¼ 5).

Fig. 1. Appearance characteristics(A), reproductive organs examination (B and C), and semen of the MSTN/ boar(D).

Item

WT

MSTN/

VCL (um/s) VSL (um/s) VAP (um/s) ALH (um) LIN (%) WOB (%) SRT (%)

95.59 ± 2.03 41.27 ± 0.60 53.52 ± 0.55 4.19 ± 0.04 43.29 ± 1.40 56.11 ± 1.51 77.11 ± 0.64

99.05 ± 2.77 42.97 ± 0.66 55.83 ± 1.06 4.19 ± 0.09 43.50 ± 1.30 56.47 ± 1.29 77.09 ± 2.15

Values are expressed as the mean ± SEM of five replicates.

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Fig. 3. IVF blastocysts induced from MSTN/ boar semen. Scale bar: 100 mm.

Fig. 2. Assessment of the semen quality of the MSTN/ and control boar semen using sample staining. Semen morphology staining, A: (a) normal semen, (b, c) abnormal head, (d) plasma drops, and (e) abnormal tail. Semen acrosome state staining, B: (f) intact and (g) reacted acrosome. Hoechst 33342, PI, and JC-1 staining of boar spermatozoa, C: (h) live semen cells (Hoechst33342 positive and PI negative), with an intact plasma and mitochondrial membrane integrities, exhibit blue fluorescence over the head and intense orange-red fluorescence over the midpiece, with a high mitochondrial transmembrane potential, (i) live semen cells, with an intact plasma membrane integrity and low mitochondrial transmembrane potential, show blue fluorescence over the head and green on the midpiece, (j) dead semen cells with a damaged plasma membrane show red fluorescence over the head and display less green or no green fluorescence over the midpiece. Scale bar: 20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

fertility evaluation. Attention should be given to the body condition and conformation. In the current study, there were not any abnormalities in the head, back, or abdomen (Fig. 1A). Kieffer and Cartwright reported a higher incidence of underdeveloped external genitalia. Additionally, 4e7% of young BB breeding bulls had a persistent frenulum of the penis [8,9]. However, in this study, there was nothing abnormal with the scrotum, prepuce, or penis of the MSTN/ boar (Fig. 1B and C). Libido is an important feature of boars, but there is no standard procedure for the assessment of their sexual behavior. The libido evaluation is performed on the basis of the total time that elapsed from the entrance into the semen collection room to the end of the ejaculation [10]. A boar's libido performance was positively correlated with the serum content of testosterone and estradiol-17 b [11,12]. Boar libido was evaluated with reference to a standard that has been previously reported: (1) the time from when the boar entered the collection area until it mounted the semen-collection dummy and started ejaculating, and (2) the duration of the ejaculation [11]. These measurements were recorded for each ejaculate throughout the sampling period. In the current study, the total time of the MSTN/ boar was shorter than that of the control boar

Table 3 Detailed semen evaluation of the MSTN/ and control boars, using sample staining (n ¼ 9). Item

WT

MSTN/

Deformity (%) Acrosome integrity (%) Viability (%) Mitochondrial activity (%)

8.61 ± 0.69 92.95 ± 0.28 89.63 ± 0.80 84.06 ± 0.81

8.04 ± 0.83 91.42 ± 1.02 91.78 ± 1.08 84.89 ± 1.24

Values are expressed as the mean ± SEM of nine replicates.

(6.83 ± 0.23 and 7.53 ± 0.31 min, respectively), but there was no significant difference. Therefore, the libido of our MSTN/ boar was normal. The manifestation of sexual instinct in boars is more complex and requires a different definition of libido and consideration for its importance. It is quite certain that in the future this will be the subject of numerous studies. The physical parameters of boar semen are the color, odor, pH, semen volume, semen concentration, and spermatozoa motility. The semen color and odor are important physical semen parameters. Normal boar semen is milk-white in color, with bluish shadows, and has an odor of reeking milk. With regards to the pH, at point of collection, a semen pH of higher than 8 denotes a lowquality or the presence of an infectious process in the genital tract or accessory sex glands. Changes in the pH negatively influence the spermatozoa viability and motility [13,14]. The semen pH value of the MSTN/ boar was not significantly different from that of the control. The color, odor, and pH of the semen of MSTN/ boar were the same as described above. This proves that the physical parameters of the semen of the MSTN/ boar are normal. The semen concentration and volume of ejaculate are the most important factors for determining the physical semen parameters. From previous studies, we can observe that the semen volume of the MSTN/ boar was significantly lower than that of the control (168.78 ± 6.70 and 223.11 ± 21.21 mL, respectively). The concentration of the semen of the MSTN/ boar was higher than that of the control (4.53 ± 0.28  108 and 3.99 ± 0.40  108 spermatozoa/ mL, respectively), but this difference was not statistically significant. The total semen of the MSTN/ boar was lower than that of the control, but this difference was not statistically significant. Some author reported the total number of semen increased with the ejaculate volum, the differences were significant, and a directly proportional correlation (r ¼ 0.63) between the number of spermatozoa and ejaculate volume was found. The higher the semen concentration, the smaller the ejaculation volume, but no significant difference was found [15,16]. A previous study showed that the semen concentration is usually the negative of the ejaculation volume [17], which was similar to our results. The previous study explains that the lower volume means a lower total semen and higher concentration of the semen in the MSTN/ boar. Spermatozoa motility is considered as being one of the most important stages of semen examination. Motility was also the parameter used to evaluate the thermo-resistance of boar spermatozoa. A high motility potentially correlate with the mitochondrial activity and viability, as the mechanisms involved in the control of the spermatozoa motility is the modulation of their mitochondrial activity, which is absolutely necessary to maintain the overall semen motility [18,19]. Mitochondria in mature semen

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Table 4 Comparison of the developmental capacity of IVF embryos induced with the semen of the MSTN/ and control boars (n ¼ 3). Item

No. of oocytes

Cleavage rate (%)

Blastocyst rate (%)

No. of blastocyst cells

WT MSTN/

254 247

65.2 ± 1.6 69.1 ± 0.7

22.8 ± 1.4 20.2 ± 1.2

42 ± 7 46 ± 7

The cleavage rate and blastocyst rate were evaluated at days 2 and 7, respectively. Values are expressed as the mean ± SEM of three replicates.

have a condensed morphology with a compacted matrix, allowing for a greater efficiency of energy production. The energy demand for the motility apparatus of spermatozoa requires a higher level of respiratory activity, a lack of which may result in metabolic disorders [20,21]. In this study, the rate of the motility and activity of the mitochondria in MSTN/ boars was not significantly different from that of the control. Additionally the rate of the activity of the mitochondria is similar with that who reported that the mitochondria activity of the Large White breed [22]. The plasma membrane integrity of fresh semen was 85.1 ± 4.8% and 77.8 ± 1.3% in the Landrace and Large white breeds respectively [21,22]. Hoflack et al. reported that a high proportion of ejaculated BB spermatozoa is structurally damaged or dead. This higher proportion of structurally damaged (or dead) spermatozoa, results in a significantly lower percentage of motile spermatozoa compared to HF bulls [5]. However, in the current study, the plasma membrane of the semen of the MSTN/ boars was not significantly different from that of the control. Among the many traits of semen, the percentage of normal spermatozoa is related to the fertility. The presence of semen abnormalities (such as an abnormal head, damaged acrosomes, midpiece defects, and proximal/distal droplets on the tails) is generally more common in the BB breed [5], and these abnormalities (in particular the abnormal midpieces, tails, droplets) might negatively influence the semen velocity [23]. In this study, our result was contrary to the previous study, as the abnormalities of the spermatozoa in the MSTN/ boar were not significantly different compared with those of control. The morphology of the acrosome is an extremely important criteria for spermatozoa evaluation, because of the localization of enzymes in this structure that take part in fertilization process [24]. Ejaculates are considered as low quality when more than 25% of the semen show acrosome abnormalities. In this study, the acrosome integrity in the MSTN/ boar was not significantly different from that of the control (91.42 ± 1.02 and 92.95 ± 0.69, respectively). In swine, the penetration of oocytes by more than one spermatozoon affects approximately 50% of in vitro fertilized oocytes. Consequently, the in vitro production of pig embryos from genetically superior animals or endangered breeds is hampered [25,26]. The fertilizing ability of boar semen depends upon multiple factors. It is difficult to confirm the fertilization outcome based on only some of the semen quality parameters. Therefore, the assessment of the total fertilization efficiency, embryo cleavage rate, and blastocyst rate is of the utmost importance [27]. Beek et al. reported

that the quality of the semen has a significant influence on IVF [28]. Our experimental results agree with this. In the current study, the important semen quality parameters of the MSTN/ boar were not significantly different from those of the control. The IVF results show that the rates of cleavage and blastocyst were not significantly different compared with those of the control. Arthur et al. reported that the DM phenotype could reduce fertility and it has been suggested that DM embryos have a higher mortality rate [29]. In this study, nine F1 offspring were successfully bred through artificial insemination and the F1 generation was healthy. In this experiment, the semen quality of the MSTN/ boar and in vitro fertilization results (fertilization and cleavage rates) were not significantly different compared with those of the control group. However, the MSTN/ litter size after artificial insemination was less than that of the control (the control group was from the same swine farm). Recently, a positive effect for MSTN on fertility was determined. After IVF, when MSTN was inhibited, the rate of blastocysts was significantly reduced [30]. This may be related to the low number of offspring in this experiment. In fact, we were training three MSTN/ boars for semen collection. However, due to double muscling phenotype of MSTN/ boars, it was difficult for them to mount the sows. As a result, we collected semen from only one MSTN/ boar, which was used to fertilize only two sows. To decipher the reason for the smaller litter size, long-term and large sample tests are required. 5. Conclusions The sexually matured adult homozygous MSTN mutant boar has an obvious ‘double muscling’ phenotype and low backfat thickness. The MSTN/ boar has the advantages of more muscle and less fat. The MSTN/ boar has a normal reproductive organ, sexual desire, semen quality, and reproductive performance. In this study, it was demonstrated that the MSTN/ boar can produce healthy offspring and it can be used as the terminal male parent. We expect that the MSTN/ boar will be developed into new super lean meat varieties in the future. Competing interests The authors declare that they have no competing interests. Authors' contributions SZH performed sperm quality assessing and in vitro fertilization with manuscript writing. SSJ performed MSTN/ boar health checks, body measurements, and artificial insemination. QG and ZBL collected semen samples. MFX and WJX contributed to care and delivery of animals. JDK and XJY contributed to experimental design. All authors read and approved the final manuscript. Acknowledgements

Fig. 4. Artificial insemination piglets from MSTN/ boar semen. (A) 24 h postfarrowing, and (B) 24 days post-farrowing.

This work was supported by the Science and Technology Development Project in Jilin Province (20170204035N Y) and by the National Natural Science Foundation of China (No. 31860297).

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