The Administration Effect of Lactic Acid Bacteria Reducing Environmental Alkyl and Fatty Acid Hydroperoxides on Piglets in the Absence of Antimicrobial Agents and in the Presence of Iron

A lactic acid bacterium, Lactiplantibacillus plantarum P1-2 (LpP1-2), can reduce environmental fatty acid hydroperoxides. The administration of LpP1-2 to oxygen-sensitive short-lived nematode mutants and iron-overloaded rats reduced the oxidative stress-related index. Since young piglets have a weak defense system against oxidative stress and are vulnerable to environmental stress, antimicrobial agents have been administered in the rearing. Based on these results, we investigated the effect of LpP1-2 administration to prepartum sows and infant piglets until weaning without antimicrobial agents on the growth of young piglets. The group including both sows and piglets that were administrated with lactic acid bacteria containing LpP1-2 (LABLp) until the end of lactation showed the growth-promoting effect of piglet from lactation to early regular rearing, and even in late regular rearing. Blood biochemical markers were in the normal ranges in both LABLp-administrated and non-administrated groups, but various disease-related markers tended to decrease in the administrated group. To investigate the effects of LABLp administration on postpartum piglets, the piglets born from prenatally LABLp-administrated sows were divided into two groups and then administrated with or without LABLp. The piglets in the LABLp-administrated group tended to grow very slightly higher than those in the non-administrated group from lactation to early regular rearing. After that, the growth in both groups was almost the same. These results suggest that LABLp administration to prepartum sows is essential for the growth-promoting effect. The postnatal LABLp-administrated piglets showed a lower serum lipid peroxidation index than the non-administrated piglets, and had higher numbers of lactic acid bacteria and bifidobacteria in feces at the end of LABLp treatment. In fattening performances, the LABLp-administrated group showed a significant improvement in meat quality. We also discuss the growth and physiological effects by LpP1-2 administration with iron on piglets because iron administration is another important issue in piglet farming.


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Antimicrobial resistance (AMR) infections are widespread worldwide [1,2], and to prevent 50 AMR, it is essential to limit the use of minimum antimicrobial agents to the minimum necessary 51 and to prevent the release of them into the environment. There are two types of antimicrobial 52 agents for livestock: those that are mainly used for the treatment of diseases [3] and those that are 53 mainly used for the growth promotion of livestock [4]. The addition of both antimicrobial agents 54 to livestock feed has been in common, making livestock indigenous bacteria drug resistant. As a 55 result, it is concerned that their release into the environment may lead to horizontal transmission 56 to humans. To prevent this, risk management measures for antimicrobial feed additives have been 57 carried out [5].
In pig growth, young piglets are sensitive to environmental stress caused by heat and temperature 59 changes [6-8], and antimicrobial agents have been administrated to young pigs to prevent disease and 60 promote growth [3,4]. In young piglets, it is known that oxidative stress index increases due to 61 environmental stress [8]. Since oxidative stress is a cause of various diseases, enhancement of the 62 defense system against oxidative stress in young piglets is expected to prevent the diseases and 63 promote growth during the juvenile stage. 64 The authors isolated a lactic acid bacterium, (LpP1-2), from fermented food. This strain reduces 65 environmental alkyl hydroperoxides and fatty acid hydroperoxides and converts both peroxides to the 66 less toxic hydroxy form via two electron reduction [9]. Oral administration of LpP1-2 to the oxygen-67 sensitive short-lived nematode mutant resulted in a significant expansion of its lifespan, suggesting that 68 LpP1-2 inhibits internal oxidative stress [9]. To specify the organs involved in this response, we 69 performed a similar experiment on iron-overloaded rats in which lipid peroxidation was induced. The 70 administration of LpP1-2 showed a significant reduction in the peroxidation index value in the colonic 71 mucosa of these rats [9]. In this report, we examined about the effect of LpP1-2 administration to 72 prepartum sows and postpartum piglets for the purpose of enhancing the defense power in young 73 piglets against oxidative stress.

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In piglet rearing, iron supplements are administered to piglets to prevent iron deficiency anemia 75 during the growth process [10][11][12]. However, since iron is a factor that causes the Fenton reaction 76 [13], piglets with immature oxidative stress defense systems may exhaust energy to cope with 5 77 such stress. In this report, to develop a probiotic material to replace antimicrobial agents, we 78 created an antimicrobial-free feed supplemented with iron and LpP1-2, and investigated the effect 79 of LpP1-2 on the growth of young piglets.

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Test animals 83 In Test I, two sows of WL breed (Yorkshire -Landrace) born in January 2009 were used as test 84 sows and were synchronized to have the same farrowing date. One sow (α) was given lyophilized 85 LABLp powder from 28 days before farrowing until weaning, and the other one (β) was given 86 skimmed milk powder as a placebo (Fig 1). The sows were artificially inseminated with Duroc semen 87 and the piglets born (27 in total) were used as test piglets. A mother-child exchange was made for half 88 of the newborn piglets. The piglets were weaned 28 days after farrowing. Lyophilized LABLp powder 89 was administered to the piglets suckled by the sow α (14 piglets, LAB group, piglets A (n=7) and 90 piglets C, (n=7)) from immediately after delivery until weaning. Skimmed milk powder was 91 administered to the piglets suckled by the sow β (13 piglets, Control group, piglets B (n=6) and piglets 92 D, (n=7)) instead of lyophilized LABLp powder. No antimicrobial agents were added to the diets of 93 either sow α or β. We monitored the weights gain and blood biochemistry of the piglets during rearing.

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In Test II, as in Test I, one WL (Yorkshire -Landrace) sow was artificially inseminated with 95 Duroc semen, and the piglets (10 piglets) born were used as test piglets (Fig 2). The piglets were 6 96 divided into two groups, half of which received lyophilized LABLp powder immediately after 97 delivery until weaning (LAB group), and the other half were administered skimmed milk powder 98 instead of lyophilized LABLp powder (Control group). The administration of LABLp to the sow 99 and the weaning were the same as the sow α in Test I. The test period was from piglet farrowing 100 to the end of fattening in both Tests I and II.

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In Test II, the serum lipid peroxidation index, body weight gain, and fecal microbial flora of 102 piglets were investigated. Piglets of finishing phase were examined for body weight, branch 103 weight at slaughter, yield, back fat thickness, and meat quality when they were shipped at 157 104 days of age.

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Test feed 107 Each sow was fed 2.5 kg/day of sow formula (Big Mama, Toyohashi Feed Co., Ltd., Japan) 108 containing no antimicrobial agents before farrowing and 5.0 kg/day after farrowing. The sow α 109 was administrated 25 g of lyophilized LABLp before farrowing, and 50 g after farrowing before 110 feeding. The sow β received 25 g of skimmed milk powder as a placebo before farrowing and 50 111 g after farrowing before feeding, instead of lyophilized LABLp powder. A homemade diet (Table   112 S1) given to the piglets were gradually increased from birth to 56 days of age. After birth, 1 g of 113 lyophilized LABLp dispersed in 10 ml of pure water per piglet was administrated to piglets A and 114 C in the LAB group (Fig 1). In the control group of piglets B and D, 1 g of skimmed milk powder 7 115 as a placebo was administered instead of the lyophilized LABLp powder (Fig 1). For iron 116 supplementation, 800 mg/ml of iron dextran (equivalent to 200 mg/ml of iron, Iron Syrup S, 117 Scientific Feed Laboratory Co., Ltd., Japan) was orally administrated at a dose of 1 ml/head on 118 the third day after birth. Other rearing management followed the conventional rearing method of    The differences between two groups were analyzed by unpaired t-test. Data obtained from three 165 or more groups were analyzed using non-repeated analysis of variance (non-repeated ANOVA).

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When the result of non-repeated ANOVA was significant, the Student-Newman-Keuls method 167 was conducted. P-values of 0.05 or less were considered to have a significant difference. In two littermate sows (α and β) synchronized delivery, the sow (α) was administrated with 172 lyophilized LABLp powder from 4 weeks before farrowing until weaning (28 days after farrowing).

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On the other hand, the sow (β) was administrated with skimmed milk powder (Fig 1). 174 Thirteen piglets (piglets A and C) were born from the sow α and 14 piglets (piglets B and D) from 175 the sow β (Table S2). To evaluate the effect of LABLp administration before and after parturition, half 176 of piglets born from each sow were suckled by mother sow exchange. Mother sow α fed 7 piglets born 177 from her (piglets A) and 7 piglets born from mother sow β (piglets C). Mother sow β fed 7 pigs born 178 from her (piglets D) and 6 pigs born from mother sow α (piglets B). Piglets A and C were administrated  Table S2).

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From the lactation period (28 days after birth) to the early normal rearing period (28 to 56 days of 183 age), the weight gain after 7 days of age in Group A (piglets A) was higher than that in Group D 184 (piglets D), and even in the late normal rearing (56 to 180 days of age) (Fig 3). The weight gain in 185 Group B (piglets B) was lower than that in Group D (S1 Fig). The weight gain in Group C (piglets 186 C) was almost the same as that in Group D (S1 Fig). In Groups A and D, the mother-child system 187 at birth was continued, while in Groups B and C, mother-child exchange was carried out after 11 188 delivery. The difference in weight gain observed between Groups B and C might be due to the 189 stress on the piglets caused by mother-child exchange. 190 Subsequently, to see the effect of LABLp on the growth of piglets, a biochemical comparison 191 of the blood of piglets A (the offspring of sow α, LABLp administration before and after weaning) 192 and D (the offspring of sow β, non-administration before and after weaning) without mother-child 193 exchange was carried out.

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In both groups, all 24-test items remained within the normal range, regardless of the presence 195 or absence of LABLp administration (Fig 4, S2 Fig, S3 Fig). The tests in which significant 196 differences were observed between the two groups are described below.

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The number of red blood cells and hematocrit values were significantly higher in the LABLp -198 administrated (administration) group than in the non-administrated (control) group at 56 days of 199 age. On the other hand, the white blood cell count, which is a marker that increases with bacterial 200 infection, was significantly higher in the administration group than in the control group at 28 days 201 of age. However, it reversed at 56 days of age, and the control group showed higher values.

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ALP is a marker for liver, hepatic pathway, small intestine, and bone disorder, total bilirubin 203 level is a marker for liver disorders, and BUN is a marker for renal disorders. ALP was higher at 204 in the control group than in the administration group at 28 days of age. Total bilirubin and BUN 205 were higher in the control group than the administration group at the age of 56 days.

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In Group A, in which the mother sow was administrated with LABLp before and after delivery, a 207 growth promoting effect on the piglets was observed not only during the lactation period but also 208 during the early normal rearing period (28-56 days of age) after the end of administration (Fig 3). The 209 detection values of the above disease markers in the blood during the normal rearing period were lower 210 in the administration group than in the control group. The growth-promoting tendency in the 211 administration group was also observed during the late normal rearing period (56-180 days of age) 212 (Fig 3).

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Test II 215 The following experiment was conducted to examine the effect of LABLp administration after 216 delivery. To avoid the stress caused by mother-child exchange, piglets born from the mother sow (sow 217  in Fig 2) which was administrated with lyophilized LAB powder before delivery were divided into 218 two groups, LABLp administrated group (Piglets F, in Fig 2) and non-administrated group (Piglets E, 219 in Fig 2), and the effect of LABLp administration after delivery was investigated (Fig 2, Table S2).

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The growth of piglets before and after weaning in the LABLp -administrated group F tended to 221 increase slightly more than that in the non-administrated group E, but it was not significant, and then 222 the growth in both groups was almost the same (Fig 5). The change in body weight in both groups was 223 almost the same as that of the LABLp-administrated group (LAB in Fig 3) consisting of the piglets 224 born and raised from the sow α in Test I.

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The lipid peroxidation index in the serum of the LAB administrated group F was lower than 226 that of the control group E at both 28 and 56 days of age (Fig 6). The numbers of both lactic acid 227 bacteria and bifidobacteria in feces were high at weaning (28 days of age) after completion of 228 LABLp administration. The number of lactic acid bacteria count was also high in the early 229 weaning period (56 days of age) after completion of LABLp, but the number of bifidobacterium 230 count was the same as that of the control group at 56 days of age. The number of Clostridium 231 perfringens was not different between the two groups and was below the detection limit at 56 days 232 of age (Fig 7). After finishing normal rearing (26 weeks of age), in piglet fattening performance 233 the LAB-administrated group showed better results than those of the non-administrated group in 234 terms of carcass weight, yield, grade, and significant improvement in meat quality (Table 1).

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The LABLp-administrated group of mother sow and her delivered piglets showed slightly 237 higher growth promotion (weight gain) in the young piglets than the non-administration group of 238 those. In addition, the piglets born from the LABLp-administrated mother pig had a similar

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In this study, we found a decrease in lipid peroxidation index by TBARS assay of blood collected 259 from LAB-administrated piglets. Previously, we showed that the reduction of lipid peroxidation index administration may have suppressive effect on iron toxicity. Previously, we showed that LpP1-2 296 must be administered as viable bacteria to exert this activity effectively [9]. The present study 297 demonstrated its effectiveness in pigs. The number of LpP1-2 found in the feces of the LAB-298 administrated group showed a higher than that of the non-administrated group (Fig 7). However, 299 the present study did not directly prove the presence or absence of LpP1-2 in the feces during 300 normal times. Analysis of the intestinal flora will be an issue for the future.
17 301 We expect that the inhibitory effect of lactic acid bacteria on the toxicity of administered iron 302 observed in this study could be applied not only to reduce iron toxicity in pigs but also to inhibit 303 ferroptosis. In the future, probiotics need to be verified from a wide perspective, containing 304 analysis of the mechanism by which lactic acid bacteria such as LpP1-2 suppress iron toxicity.      Fig. 1). The data is expressed as the mean value ± standard deviation (SD).

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Double asterisks (**) indicate a significant difference between the two groups (p < 0.01, 456 unpaired t-test). The inset shows the weight change during the entire rearing period.    Fig. 1). Also, the dotted 500 line with white-square is the average weight of piglets B (Control group (n=6) in Fig. 1) and 501 dotted line with white triangle is the average weight of piglets D (Control group (n=7) in Fig. 1).

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The inset shows the weight change during the entire rearing period. 503 504 S2 Fig and S3 Fig. Blood biochemistry, blood cell counting of piglets in Test I. 505 The gray bar indicates the average value of piglets A (LAB group (n=5) in Fig. 1), and the white 506 bar indicates the average value of piglets D (Control group (n=5) in Fig. 1). The data is 507 expressed as the mean ± standard deviation (SD). 508 29 509 Table S1. Composition of homemade diet. 510 The composition of a homemade diet is shown as a percent. The homemade diet given to the 511 piglets in Test I and Test II gradually increased from birth to 56 days of age.  Table S2. Body weight changes of piglets in Test I and Test II. 514 Weighing results for individual piglets in Tests I and II are shown. The weight record for piglets 515 that died during the rearing process remains blank.