Probiotics or Synbiotics Addition in Bama Mini-pigs’ Diets Improves Growth Performance, Carcass Traits, and Meat Quality by Altering Plasma Metabolites and Related Gene Expression of Offspring Pigs

Qian Zhu Institute of Subtropical Agriculture Chinese Academy of Sciences Mingtong Song Institute of Subtropical Agriculture Chinese Academy of Sciences Md. Abul Kalam Azad Institute of Subtropical Agriculture Chinese Academy of Sciences Cui Ma Institute of Subtropical Agriculture Chinese Academy of Sciences Yulong Yin Institute of Subtropical Agriculture Chinese Academy of Sciences Xiangfeng Kong (  nnkxf@isa.ac.cn ) Institute of Subtropical Agriculture Chinese Academy of Sciences https://orcid.org/0000-0001-64066445


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Microbes inhabited in the gut have strong metabolic activity and play critical roles in 34 regulating the growth and development, nutrient metabolism, immunity response, and 35 health of the host [1]. Previous studies have shown that the intestinal microbiota can 36 affect muscle growth, nutrient metabolism, and muscle fiber type transformation [2], 37 indicating that intestinal microbiota can influence the carcass traits and meat quality 38 through multiple metabolic pathways. Increasing evidence has shown that maternal 39 probiotics supplementation has beneficial effects on offspring piglet's growth and 40 development. For example, dietary Bacillus addition during late gestation and lactation 41 has beneficial effects on growth performance and intestinal microbiota modulation of 42 piglets [3]. In addition, dietary prebiotics such as xylo-oligosaccharides (XOS) 43 intervention could also facilitate the proliferation of beneficial microbes and thus 44 improve the growth performance of weaned piglets by regulating intestinal health [4]. 45 However, it has remained unknown how maternal gut microbiota regulates the meat 46 quality of offspring and whether the gut microbiota or their metabolites have positive 47 effects on meat quality. Therefore, maternal gut microbiota intervention through 48 probiotics, prebiotics, and synbiotics addition would be used as a strategy to improve 49 the meat quality of the offspring. 50 Maternal health and nutritional status during gestation and lactation play a vital 51 role in the growth and development of the fetuses and have potential long-term effects 52 on offspring pigs. Studies have demonstrated that changing maternal nutrition during 53 muscle compared with the control group. Moreover, serine (Ser) and His contents in 284 the LT muscle were higher (P < 0.05) in the synbiotics group compared with the other 285 three groups at 125-day-old. 286 Fatty acid contents of longissimus thoracis muscle 287 The effects of dietary probiotics and synbiotics addition in sow diets during pregnancy 288 and lactation on amino acid contents in the LT muscle of offspring pigs are shown in 289 Table 5. At 65-day-old, compared with the control group, the contents of C18:2n6c and 290 PUFA in the antibiotic and probiotics groups and the contents of C10:0, C12:0, and 291 C14:0 in the synbiotics group were increased (P < 0.05) in the LT muscle of pigs, 292 whereas the contents of C16:0 and C20:1 in the probiotics group were decreased (P < 293 0.05) in the muscle of pigs. In addition, the content of C24:0 was increased (P < 0.05) 294 in the probiotics group, while the contents of C18:1n9c, C20:0, and SFA in the 295 probiotics group were decreased (P < 0.05) compared with the other three groups. 296 Moreover, the content of C10:0 was increased (P < 0.05) in the synbiotics group 297 compared with the other three groups, and the content of IMF was decreased (P < 0.05) 298 in the probiotics group compared with the antibiotic and synbiotics groups. At 95-day-299 old, the content of C10:0 in the probiotics and synbiotics groups and the content of 300 C14:0 in the probiotic group were increased (P < 0.05) in the LT muscle of pigs, while 301 the content of C20:1 in the antibiotic and synbiotics groups was decreased (P < 0.05) 302 compared with the control group. In addition, the content of C12:0 was increased while 303 the content of C16:1 was decreased in the antibiotic, probiotics, and synbiotics groups 304 compared with the control group (P < 0.05). Moreover, the contents of UFA and MUFA 305 were increased (P < 0.05) in the probiotics group compared with the other three groups 306 at 95-day-old. At 125-day-old, the contents of C15:0 in the probiotics and synbiotics 307 groups, C17:0 in the probiotics group, C18:2n6c in the synbiotics group, and C18:1n9t 308 in the antibiotic, probiotics, and synbiotics groups were decreased in the LT muscle of 309 pigs compared with the control group. In addition, the contents of C16:1 in the 310 probiotics group and IMF in the synbiotics group were increased (P < 0.05) compared 311 with the other three groups. 312 The mRNA expression of myosin heavy chain isoforms, myogenic regulatory 313 factors, and lipid metabolism related genes in longissimus thoracis muscle 314 The effects of probiotics and synbiotics addition in sow diets on mRNA expression of 315 MyHC isoforms, MRFs, and lipid metabolism related genes in the LT muscle of 316 offspring pigs at 65-, 95-, and 125-days are shown in Figure 1. Compared with the 317 control group, the mRNA expression of MyHC IIa in in the antibiotic, probiotics, and 318 synbiotics groups was up-regulated (P < 0.05), while the mRNA expressions of MyHC 319 IIx and MyHC IIb in the probiotics group were down-regulated (P < 0.05) in the LT 320 muscle of pigs at 65-day old (Figure 1 A−B). Moreover, the mRNA expression of 321 MyHC I was up-regulated (P < 0.05) in the probiotics group compared with the other 322 three groups and the mRNA expression of Myf6 was down-regulated (P < 0.05) in the 323 antibiotic group compared with the probiotics group at 65-day-old (Figure 1 A−B). At 324 95-day-old, the mRNA expression of MyHC IIb and Myf5 in the LT muscle were up-325 regulated (P < 0.05) in the probiotics and synbiotics groups compared with the control 326 and antibiotics groups. The mRNA expression of MyHC I and Myf6 in the LT muscle 327 were up-regulated (P < 0.05) in the synbiotics group, while the expression of MSTN 328 was down-regulated (P < 0.05) when compared with the other three groups (Figure 1  probiotics, and synbiotics groups compared with the control group, whereas the mRNA 340 expression of lipoprotein lipase (LPL) was up-regulated (P < 0.05) in the probiotics 341 group compared with the other three groups. At 95-day-old, the mRNA expression of 342 LPL in the antibiotic, probiotics, and synbiotics groups and the mRNA expression of 343 acetyl-CoA carboxylase (ACC) in the probiotics group were up-regulated (P < 0.05) 344 compared with the control group. Moreover, the mRNA expression of fatty acid 345 synthase (FASN) in the probiotics and synbiotics groups and the mRNA expression of 346 SCD in the synbiotics group were up-regulated (P < 0.05), compared with the control 347 and antibiotic groups. At 125-day-old, the mRNA expression of LPL was up-regulated 348 (P < 0.05) in the antibiotic, probiotics, and synbiotics groups compared with the control 349 group. The mRNA expression of ACC was up-regulated in the antibiotic and synbiotics 350 groups and down-regulated in the probiotics group compared with the control group. 351 Moreover, the mRNA expressions of FASN and SCD were up-regulated (P < 0.05) in 352 the antibiotic group compared with the other three groups, while the mRNA expression 353 of hormone-sensitive triglyceride lipase (LIPE) was down-regulated compared with the 354 control group. 355

Plasma biochemical parameters 356
To evaluate the effects of maternal probiotics or synbiotics addition in sows' diets on 357 offspring pigs, we measured the plasma biochemical parameters at 65-, 95-, and 125-358 day-old, and the results are presented in Table 6. At 65-day-old, compared with the 359 control group, the plasma activity of α-AMS in the antibiotic, probiotics, and synbiotics 360 groups, while the plasma activity of LDH was increased in the antibiotic group and 361 decreased in the probiotic group (P < 0.05). The concentrations of LDL-C and TC were 362 increased in the antibiotic group while decreased in the synbiotics group compared with 363 the control group (P < 0.05). In the synbiotics group, the concentration of HDL-C was 364 increased compared with the other three groups, while the concentration of Glu was 365 decreased compared with the control and probiotics groups (P < 0.05). Moreover, the 366 concentrations of ALB in the antibiotic group and CHE in the probiotics group were 367 decreased (P < 0.05) compared with the other three groups. At 95-day-old, the plasma 368 activities of AST and LDH in the antibiotic and probiotics groups, the concentration of 369 GLU in the antibiotic, probiotics, and synbiotics groups, and concentration of TG in the 370 synbiotics group were decreased (P < 0.05) compared with the control group. 371 Compared with the control and antibiotic groups, the plasma CHE activity and UN 372 concentration were decreased (P < 0.05) in the probiotics and synbiotics groups. 373 Moreover, the plasma concentration of AMM was decreased (P < 0.05) in the probiotics 374 group compared with the other three groups. At 125-day-old, the plasma concentration 375 of HDL-C was increased (P < 0.05), whereas the AST and LDH activities were 376 decreased (P < 0.05) in the antibiotic and probiotics groups compared with the control 377 group. The plasma concentration of AMM was decreased (P < 0.05) in the antibiotic, 378 probiotics, and synbiotics groups, while the plasma activity of α-AMS was increased 379 (P < 0.05) in the antibiotic and synbiotics groups, when compared with the control 380 group. Moreover, the plasma concentration of TP in the antibiotic group was decreased 381 (P < 0.05), and the plasma concentration of TG in the synbiotics group was increased 382 (P < 0.03) compared with the other three groups. 383 Plasma free amino acid concentration 384 Table 7 presents the effects of maternal probiotics or synbiotics addition in sow's diets 385 in offspring pigs' plasma free amino acid concentration. At 65-day-old, the plasma 386 histidine (His) concentration in the probiotics group, the hydroxy-proline (Hypro) 387 concentration in the antibiotic, probiotics, and synbiotics groups, and the β-388 aminoisobutyric acid (β-AiBA) concentration in the antibiotic and synbiotics groups 389 were increased (P < 0.05), whereas the plasma Gly, Ile, sarcosine (Sar), and taurine 390 (Tau) concentrations in the antibiotic and probiotics groups and the Leu concentration 391 in the probiotics group were decreased (P < 0.05), compared with the control group. 392 Compared with the control and antibiotic groups, the plasma citrulline (Cit) and 393 cystathionine (Cysthi) concentrations were increased (P < 0.05) in the synbiotics group, 394 while the plasma alanine (Ala) concentration was decreased (P < 0.05) in the probiotics 395 group. The plasma γ-amino-n-butyric acid (γ-ABA) concentration was increased in the 396 synbiotics group compared with the other three groups, while the plasma tyrosine (Tyr) 397 concentration was decreased and the plasma hydroxy-lysine (Hylys) was increased in 398 the probiotics group compared with the other three groups (P < 0.05). However, the 399 antibiotic group had increased (P < 0.05) plasma threonine (Thr) and Pro concentrations 400 compared with the other three groups. 401 At 95-day-old, the plasma Ile, Leu, Cit, Tau, α-aminoadipic acid (α-AAA), α-ABA, 402 β-alanine (β-Ala), and γ-ABA concentrations were decreased, and the plasma γ-amino-403 n-butyric acid (β-AiBA) was increased in the antibiotic, probiotics, and synbiotics 404 groups, compared with the control group (P < 0.05). In addition, compared with the 405 control group, the plasma -AiBA concentration was increased, and the ethanolamine 406 (EOHNH2) concentration was decreased in the probiotics and synbiotics groups (P < 407 0.05). Compared with the control and antibiotic groups, the plasma Sar concentration 408 was increased, and the EOHNH2 was decreased in the probiotics and synbiotics groups, 409 as well the plasma Cysthi concentration was increased in the synbiotics group (P < 410 0.05). Moreover, the plasma Hylys concentration was increased, and the plasma 411 anserine (Ans) concentration was decreased in the synbiotics groups, compared with 412 the other three groups. However, the antibiotic group had increased (P < 0.05) plasma 413 1-Methyl-histidine (1-Mehis), EOHNH2, and ornithine (Orn) concentrations compared 414 with the other three groups. 415 At 125-day-old, the plasma Ans and methionine (Met) concentrations in the 416 antibiotic and probiotics groups, the plasma valine (Val) and Leu concentrations in the 417 antibiotic and synbiotics groups were increased, compared with the control group. The 418 plasma α-ABA concentration was increased, whereas the Orn concentration was 419 decreased in the antibiotic, probiotics, and synbiotics groups compared with the control 420 group (P < 0.05). Compared with control and antibiotic groups, the plasma Cysthi 421 concentration was increased in the synbiotics group, while the plasma Ser concentration 422 was decreased in the probiotics and synbiotics groups (P < 0.05). Moreover, the plasma 423 Asp and His concentrations were increased in the probiotics group, and the plasma 424 EOHNH2 concentration was decreased in the synbiotics group, when compared with 425 the other three groups (P < 0.05). However, the antibiotic group had increased (P < 426 0.05) plasma Thr, Ala, Arg, Gly, Pro, Ser, Tau, Tyr, and β-AiBA concentrations 427 compared with the other three groups. 428

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There is growing scientific and industrial interest for prebiotics, probiotics, and 430 synbiotics addition in pig production [21,22]. Numerous studies showed that dietary 431 probiotics or synbiotics could improve the health status and production performance of 432 pigs by modulating intestinal microbiota [23,24]. Therefore, the present study aimed 433 to determine the effects of maternal gut microbiota intervention via dietary probiotics 434 or synbiotics addition on the growth performance, carcass traits, meat quality, and 435 metabolites of their offspring pigs. Lactobacillus plantarum ≥ 1.0 × 10 8 CFU/g, Saccharomyces cerevisiae ≥ 0.2 × 10 8 449 CFU/g and XOS) addition can alter the abundance, diversity, and composition of gut 450 microbiota in pregnant and lactating sows [10]. However, the present study also found 451 that maternal antibiotic addition significantly decreased the ADFI of offspring pigs 452 during 66−95 day-old and increased F/G during 96−125 day-old, which may be related 453 to the adverse effects of antibiotic use during pregnancy on the offspring [28]. A 454 previous study also indicated that maternal antibiotic addition could alter the maternal 455 and fetus microbiome in utero and during the postnatal period through maternal-fetus 456 interaction, thereby affecting the health of offspring [29]. Therefore, the adverse effect 457 of maternal antibiotic addition on the growth performance of offspring needs to be 458 further studied. 459 Carcass traits and meat quality are the major factors that influence the meat flavor, 460 tenderness, juiciness, and overall consumer acceptance. In the present study, maternal 461 probiotics or synbiotics addition increased the backfat thickness and fat percentage of 462 offspring pigs at 125-day-old along with the antibiotic, while probiotics addition 463 increased backfat thickness at 65-day-old, suggesting that these additives could 464 improve body fat deposition and then improve the meat tenderness in the 125-day-old 465 pigs. Generally, the loin-eye area is positively related to the lean meat rate and 466 negatively related to backfat thickness. However, the present study showed a decrease 467 in the loin-eye area and lean meat rate in the synbiotics group at 125-day-old, indicating 468 that maternal synbiotics has no impact on lean meat. Previous studies also found that 469 dietary XOS addition has no significant effect on lean meat [30,31] that maternal synbiotics addition could improve the sense-impression of pork, while 484 probiotics addition has no positive effect on meat color. These inconsistent findings 485 might be related to the feeding stage and the type and dose of probiotics or synbiotics. 486 Additionally, the results of this study revealed that maternal probiotics or 487 synbiotics addition also improved the meat quality by increasing pH, water holding 488 capacity, and cooking yield and decreasing the shear force in the LT muscle of offspring 489 pigs. After slaughter, due to glycolysis, the accumulation of lactic acid in the muscle 490 leads to a decrease in pH, which is highly correlated with the drip loss and shear force 491 [35]. Drip loss can reflect the water-holding capacity of muscle and is also an important 492 factor affecting meat quality [36], while shear force is correlated with meat tenderness 493 (50). In the present study, maternal probiotics addition increased pH45min at 95-day-old 494 and cooking yield at 65-day-old, while maternal probiotics or synbiotics addition 495 increased water holding capacity at 65-day-old and decreased shear force at 125-day-496 old. However, maternal antibiotic addition showed an increased shear force of the LT 497 muscle at 125-day-old. A previous study reported that drip loss usually ranges from 2% 498 to 10% when the meat is cut into slices [37], which is consistent with the present study 499 (ranges 2.78%−6.32%). The results were also consistent with the previous studies by 500 Suo et al. (2012), who demonstrated that Lactobacillus plantarum ZJ316 addition 501 improved pH45min [38], and Zhou et al. (2010), who reported that Bacillus coagulans 502 could affect meat quality by decreasing drip loss [39]. Thus, the results of the present 503 study indicated that maternal probiotics addition could decrease the lactic acid 504 accumulation by improving muscle glycolysis, while maternal probiotics or synbiotics 505 addition could increase water holding capacity and cooking by reducing drip loss, 506 cooking loss, and shear force and thereby improve meat quality. 507 Changes in the nutrient contents of muscular tissue, especially the contents of IMF 508 and CP, could directly affect the sensory properties and nutritional values of meat [40]. 509 Moreover, the IMF is a major quality trait of meat, of which content influences the 510 consumers' perceptions of cooked pork palatability [41]. Fernandez et al. (1999) 511 demonstrated that the tenderness, juiciness, color, and flavor of meat were substantially 512 improved with the increase in IMF content [42]. In the present study, maternal 513 synbiotics addition increased the IMF content of LT muscle at 125-day-old, indicating 514 that the meat quality was improved by maternal synbiotics addition. The change in IMF 515 content of LT muscle is consistent with the change of shear force in LT muscle because 516 the IMF content has a negative correlation with the shear force [43]. Moreover, the 517 higher IMF content can improve the taste of meat [44]. 518 Amino acids play pivotal roles in the growth, development, reproduction, and 519 health of animals. Moreover, the composition and contents of amino acids in muscle 520 could represent the protein quality and nutritional value of meat [45]. In the present 521 study, dietary probiotic or synbiotics addition significantly increased the TAA contents 522 in the LT muscle at 95-day-old, suggesting that the nutritional value of pork of growing 523 pigs was improved by increasing amino acid deposition. Several AA play key roles in 524 the aroma and flavor profiles of muscle [46]. For example, Arg,Leu,Ile,Val,Phe,Met,525 and His present a bitter taste; Glu and Asp present a pleasant fresh taste; and Gly, Ala,526 and Ser present a sweet taste [47]. In the present study, maternal probiotics or synbiotics 527 addition increased several AA contents, including His, Arg, Asp, Leu, Ser, and Phe, at 528 different stages in the LT muscle. These changes could improve the flavor of LT muscle 529 of offspring pigs. A previous study also indicated that dietary Clostridium butyricum showed that LDH activity was decreased in the probiotics group at 95-and 125-day-566 old, GLU concentration was decreased in the synbiotics group at 65-day-old, and in the 567 probiotics and synbiotics group at 95-day-old. Therefore, the results indicated that 568 maternal probiotics addition improved the energy and glucose metabolism of offspring 569 pigs, while maternal synbiotics addition improved glucose metabolism. The animal procedures in the present study followed the Chinese Guidelines for Animal 686 Welfare and experimental protocols approved by the Animal Care and Use Committee 687 of the Institute of Subtropical Agriculture, Chinese Academy of Science. 688

Consent for publication 689
Not applicable. 690

Competing interests 691
The authors declared that no conflict of interest. 692 antibiotic group, probiotics group, and synbiotics group are 8, 6, 8, and 6, respectively. 983