產後奶量不足怎麼辦？王不留行的中藥泌乳效果研究與應用指南

Front Vet Sci. 2023 Nov 9;10:1284552. doi: 10.3389/fvets.2023.1284552

Supplementation of dietary semen vaccariae extracts to lactating sow diets: effects on the production performance, milk components, and gene expression related to mammogenesis

Chaohua Xu 1,2,†, Jiajun Xie 1,2,†, Fengjie Ji 1,3, Weiqi Peng 1,3, Yuzhuo Song 4, Xinping Diao 2,*, Hongzhi Wu 1,3,*

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PMCID: PMC10666067  PMID: 38026663

Abstract

This study aimed to investigate the effects of dietary semen vaccariae extracts (SVE) on the production performance, colostrum components, and relative gene expression related to mammogenesis of lactating sows. 48 pregnant sows were selected and randomly allocated into four groups, with six replicates and two sows per replicate. The first group was the control (CON), while the other groups received the same diet further supplemented with 1.5, 3.0 and 4.5 g SVE per kg (SV1, SV2 and SV3, respectively). Compared with the control group, (1) the average daily gain was increased (p < 0.05) in SV1, SV2, and SV3 during the 11–21 days and 1–21 days of lactation; (2) the serum insulin-like growth factor-1, insulin, prolactin, and estrogen contents in SV1, SV2, and SV3 were increased (p < 0.05) on the 1st and 21st day of lactation; (3) The plasma Lysine, Threonine, and Tryptophan concentrations were also higher (p < 0.05) in SV1, SV2, and SV3 on the 1st and 21st day of lactation; (4) The milk Lysine, Methionine, Threonine, and Tryptophan concentrations were higher (p < 0.05) in SV1, SV2, and SV3 on the 1st and 21st day of lactation; (5) The milk lactose ratio and milk protein content were increased (p < 0.05) in the groups treated with semen vaccariae on the 1st day of lactation, while the milkfat ratio and milk protein content were increased (p < 0.05) in SV2 and SV3 on the 21st day of lactation; (6) the immunoglobulin M, A, and G contents were increased (p < 0.05) in the groups treated with the semen vaccariae on the first day of lactation; and (7) the relative PRLR, STAT5a, FcRn, CSN2, and LALBA expressions were higher (p < 0.05) in the groups treated with the semen vaccariae on the 1st and 21st day of lactation. In this study, the optimum dosage was 3.0 g/kg semen vaccariae, which increased the average daily gain of piglets, total lactation yield, and serum hormone levels, improved the amino acid levels in plasma, and facilitated the milk quality, up-regulated the relative gene expressions in the mammogenesis.

Keywords: semen vaccariae extracts, lactating sows, production performance, colostrum components, mammogenesis

1. Introduction

The mammary gland is a complex secretory organ composed of many different types of cells, including vascular endothelial cells, fibroblasts, immune cells, and epithelial cells, unique to mammals (1, 2). During gestation, the mammary gland undergoes extensive tissue remodeling under hormonal regulation, including ductal branche proliferation, acinar bud development, and extracellular matrix remodeling (3). At the early stages of gestation, the mammary ductal epithelium proliferates and occupies the intraductal space, interstitial adipose tissue gradually disappears, and angiogenesis increases; at the middle stages of gestation, the developing ductal branches are surrounded by a network of capillaries; and at the late stages of gestation, as the mammary glands differentiate, alveoli reach their development peak, which covers the majority of the fat pad and exhibits some secretory activity as gestation approaches (4–6). During lactation, the mammary epithelial cells act as a double-layered tubular structure, discharging milk from the acinar lumen and supplying the young animals with immunoglobulins and energy, thus decreasing intestinal diseases and sustaining the survival and growth of the young animals (7). The gradual decrease in demand for milk as young animals grow and develop leads to stagnation of milk in the acinar lumen, which initiates the degenerative process, i.e., of epithelial cell apoptosis and the epithelial ductal tree remodeling (8, 9). The colostrum is crucial for the survival of newborn piglets, as the immunoglobulin of sow cannot pass the placental barrier (10, 11). The newborn piglets can only get the maternal immunoglobulin through colostrum, and all the early nutrients of piglets come from breast milk (12–14), so improving the lactation yield of sows to make piglets grow fast and healthy is also the top priority in pig production.

Semen vaccariae, the seed of Vaccaria segetalis, help improving the symptoms of women’s postpartum milk shortage and is traditionally used in East Asian countries for breast milk deficiency treatment because of its low price and good efficacy (15). However, its use in local breed sows and its effect on mammogenesis in sows have not yet been reported. We hypothesized that semen vaccariae also has the effect of promoting milk secretion and mammogenesis in gestating and lactating sows, so the objective of this study was to (a) examine the effects of semen vaccariae extracts on the production performance, milk components, and gene expression related to mammogenesis of lactating sows; (b) preliminary investigate the action mechanism of semen vaccariae on the lactation ability of sows; and (c) examine the valuable addition of semen vaccariae extracts in the feed of gestating and lactating sows.

2. Materials and methods

2.1. Experimental material

Forty eight primiparous sows of the Hainan local breed (Tunchang), with an average body weight of 100.00 ± 8.00 kg, the same genetic background and similar birth date were used.

Semen vaccariae extracts (SVE) were purchased from Shaanxi Hao Yang Biotechnology Co. The SVE active ingredient contents were determined by the liquid chromatograph and mass spectrometer in the Pony Testing International Group Company, Beijing, China. The semen vaccariae flavonoids content of SVE was 4.82%, the saponin content was 1.73%, and the vacsegoside content was 0.62%, detected by 1,260 Infinity II High-Performance Liquid Chromatograph, Agilent, Beijing, China.

Serum biochemical and colostrum immunoglobulin kits were purchased from Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China.

2.2. Experiment design and sample collection

The experiment adopted a single-factor design. Forty-eight 317-day-old primiparous sows in the third semester of pregnancy (107 days), with similar physical conditions and expected confinement period, were selected and were randomly allocated into four treatments with six replicates and two sows per replicate. The basal diets for the gestating and lactating sows were a corn-soybean meal type diet formulated according to the NRC (1998; 2012) standards for swine feeding, and the composition of the basal diets and nutritional levels are shown in Table 1. The first group was the control (CON), while the other groups received the same diet further supplemented with 1.5, 3.0 and 4.5 g SVE per kg (SV1, SV2 and SV3, respectively). Each group was fed the corresponding experimental feed from the 107th day of sow gestation, restricted to 3.0 kg per day for 3 days before the farrowing date. Every sow was fed 2.0 kg on the first day after farrowing and increased by 0.5–1.0 kg per day after that. The sow had then free access to feed and water. The piglet number was adjusted to 9–11 per lactating sow within 3 days after farrowing by cross-fostering. In the farrowing room, piglets were kept warm with thermal insulation and warming lamps.

Table 1.

Composition (kg/100 kg) of the basal experimental dietsa for gestating and lactating sows.

Ingredients

Content

Nutrient levels

Content

Corn, %

69.00

Digestible energyc, DE, MJ/kg

13.98

Wheat bran, %

3.00

Crude proteind, CP, %

16.35

Soybean meal, %

19.00

Calciumd, Ca, %

0.73

Fish meal, %

2.00

Available phosphorusd, AP, %

0.34

Soybean oil, %

2.60

Lysined, Lys, %

0.92

CaHPO4, %

0.70

Methionined, Met, %

0.26

Stone powder, %

1.00

Threonined, Thr, %

0.59

NaCl, %

0.70

Premixb, %

2.00

Total

100

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aBased on the NRC (1998; 2012) nutrient requirements for lactating sows.

bThe premix provided the following per kg of diet: VA 2000 IU, VD 200 IU, VE 45 IU, VK 0.5 mg, VB1 1 mg, pantothenic acid 12 mg, nicotinic acid 10.25 mg, VB6 3.85 mg, VB12 15 μg, folic acid 1.35 mg, biotin 0.21 mg, Mn as manganese sulfate 20 mg, Fe as ferrous sulfate 80 mg, Cu as copper sulfate 5 mg, I as potassium iodide 0.14 mg, and Se as sodium selenite 0.15 mg.

cCalculated value (NRC, 1998; 2012).

dAnalysed content.

The experiment lasted from the 107th day of gestation to 10 days after the weaning of piglets on the 21st day. The number of piglets and the weight of piglets per sow on the 1st and 21st days were recorded to calculate the average daily weight gain (ADG) of piglets at different stages or throughout the feeding period. In addition, the average daily feed intake (ADFI) of the lactating sows and the number of piglet-weaning litters (NWL) were recorded throughout the feeding period. The sows were observed in estrus interval (EI) from the fourth day after weaning, and the time between estrus was recorded. If the EI was more than 10 days, it was recorded as no estrus. The total lactation yield (TLY) during lactation was calculated using the formula (16, 17):

𝑇𝑜𝑡𝑎𝑙𝑙𝑎𝑐𝑡𝑎𝑡𝑖𝑜𝑛𝑦𝑖𝑒𝑙𝑑,𝑘𝑔=𝐴𝑣𝑒𝑟𝑎𝑔𝑒𝑑𝑎𝑖𝑙𝑦𝑤𝑒𝑖𝑔ℎ𝑡𝑔𝑎𝑖𝑛𝑜𝑓𝑝𝑖𝑔𝑙𝑒𝑡𝑠

×𝑁𝑢𝑚𝑏𝑒𝑟𝑜𝑓𝑙𝑎𝑐𝑡𝑎𝑡𝑖𝑜𝑛𝑑𝑎𝑦𝑠×𝑁𝑢𝑚𝑏𝑒𝑟𝑜𝑓𝑙𝑖𝑡𝑡𝑒𝑟𝑠×4.

About 30 mL of colostrum were collected with pulsating vacuum pump for sow milking (ZL001), shanghai, China, from the front, middle, and rear nipples of the sows within 2 h after the end of parturition and on the 21st day of lactation, then mixed well and stored at −20°C until further testing. On the 107th day of gestation, 1st and 21st days of lactation, 10 mL of blood was collected with disposable vacuum blood collection tubes from the ear vein of each sow. After resting the blood for 15 min, it was centrifuged at 3000 rpm for 15 min to obtain the serum with Multifuge X4 Pro ThermoFisher High performance centrifuge, Shanghai, China, which was future transferred into Eppendorf tubes and stored at −20°C for testing. The milk protein content (MPC), milk fat ratio (MFR), milk lactose ratio (MLR), and solids-not-fat (SNF) in milk were determined using a near-infrared reflectance spectroscopy instrument (Milk-Scan 134 A/B, Beijing, China). Prior to analysis, colostrum and milk were separated by centrifugation at 3000 × g at 4°C for 20 min. In addition, the Insulin-like growth factor-1 (IGF-1), Insulin (INS), prolactin (PRL), and estrogen (E) in serum, and the Immunoglobulin A (IgA), Immunoglobulin G (IgG), Immunoglobulin M (IgM) in colostrum were determined by the HITACHI Automatic Analyzer 3,500, Ibaraki-Ken, Japan. The assay was performed according to the kit instructions. The variation coefficients of all indexes in inter-and intra-assay were controlled within 5.00%. About 5 mL of blood (plasma) and milk were collected with disposable vacuum blood collection tubes and stored at −20°C for amino acids test by 1,260 Infinity II High-Performance Liquid Chromatograph, Agilent, Beijing, China.

On the 1st and 21st day of lactation, 4 mL lidocaine hydrochloride (2 mL:40 mg, Beijing Yimin Pharmaceutical Co., Ltd., Beijing, China) was injected into the second left papilla of the sows for local anesthesia, and 3 g of mammary tissue samples were collected using a live sampling gun (BARD® MAGNUM®, MG1522, United States) and disposable sampling needles (C.R. Bard. Inc., Coington GA, United States). The total RNA of the mammary tissue was isolated with TRIzol (Sigma, Saint Louis, MO) according to the RNA extraction method. The total RNA from each mammary tissue was reverse transcribed into cDNA using a Prime Script™ RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). The obtained cDNA was used for qPCR using a TB Green™ Premix Ex Taq™ PCR kit (TaKaRa, Dalian, China). The primer sequences are shown in Table 2, and the primers corresponding to the pig gene sequence were synthesized by Sangon (Shanghai, China). Samples were disposed of with an ABI PRISM 7500 SDS thermal cycler (Applied Biosystems, Foster City, CA, United States), and the following temperature program was used: one cycle at 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 34 s. Based on the 2−∆∆Ct method, relative gene mRNA expression was the mean of the normalized by β-actin and Glyceraldehyde-3-phosphate dehydrogenase expression, respectively. Moreover, all of the processes were performed in triplicates under RNase-free conditions. RT-qPCR products were cloned into a pMD18-T vector (TaKaRa) and sequenced by the Sanger method. The sequencing results were compared with the gene sequences in NCBI, and the genes amplified by the primers in Table 2 were verified as the target genes according to the alignment sequence results (16).

Table 2.

Primer Sequence list.

Gene

Gene name

Forward and reverse primers

Product size

Accession No.

PRLP

Prolactin receptor

F:5’-GGCTCCGTTTGAAGAACCAA-3’

67

NM_001001868.1

R:5’-GTCTTTCGCAGCTGGATTCTG-3’

LALBA

α-Lactalbumin

F: 5’-GGATTGACTACTGGTTGGCCC-3’

100

NM_214360.1

R: 5’-GGCAGAAGCAGCAAGACAGC-3’

CSN2

β-Casein

F: 5’-GGACTTGATCGCCATGAAGCT-3’

81

NM_214434.2

R: 5’-GCATTGAGTTCTTCCTTCGCTCT-3’

FCRn (PCRGT)

Neonatal Fc receptor

F: 5’-TTTGGGCCTGACTTTTGTGT-3’

61

NM_214197.2

R: 5’-AATTCCCCCCACGTTGC-3’

STAT5b

Signal transduction and activator of transcription 5b

F: 5’-GACTCTGAAATTGGTGGCATCA-3’

67

NM_214168.1

R: 5’-GATTCCAAAACATTCTTTCCTCAGA-3’

STAT5a

Signal transduction and activator of transcription 5a

F: 5’-ATCTCATCTATGTGTTTCCCGACC-3’

74

NM_214190.1

R: 5’-CGGAGCGAGCACAGGAGT-3’

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

F: 5’-GTCGGAGTGAACGGATTTGG-3’

76

NM_001206359.1

R: 5’-CAATGTCCACTTTGCCAGAGTTAA-3’

β-actin

–

F: 5’-AGGCTACAGCTTCACCACCAC-3’

95

AB618546

R: 5’-CCATCTCCTGCTCAAAATCCA-3’

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2.3. Statistical analysis

Statistical analyses were conducted using SPSS statistics software (version 20.0, International Business Machines Corporation, Armonk, NY, United States). Data were expressed as mean ± SEM. Statistical comparisons of different treatments were performed using one-way ANOVA or Welch ANOVA after the variance homogeneity test. Statistical differences among groups were assessed using Duncan’s multiple range test with dietary treatment as a fixed factor. Each replicate was considered as an experimental unit. The test results of all analyses were considered significant at p < 0.05.

3. Results

3.1. Semen vaccariae extracts improved the production performance of lactating sows

Compared with the control group, the ADG in SV2 and SV3 was increased (p < 0.05) by 10.00 and 9.50%, respectively, during the first 10 days of lactation, and it was increased (p < 0.05) by 4.15% vs. 6.86, 7.83% vs. 11.27, 8.29% vs. 11.27%, respectively, in SV1, SV2, and SV3 during the 11–21 days and 1–21 days of lactation. The TLY was 7.02, 11.70, and 11.70%, respectively, higher (p < 0.05) in the groups treated with the senmen vaccariae extracts than that in the control group, and it was 11.70% vs. 11.70% higher (p < 0.05) in SV2 and SV3 than that in SV1 (Table 3).

Table 3.

Effect of semen vaccariae extracts on the production performance of lactating sows.

Parameters

Con

SV1

SV2

SV3

p-value

ADG, g/d

1–10 d

200±6.82b

210±3.39ab

220±5.64a

219±5.23a

0.0431

11–21 d

217±4.26c

226±3.95b

234±3.53a

235±4.36a

0.0236

1–21 d

204±5.21c

218±3.29b

227±3.24a

227±4.03a

0.0328

ADFI, kg/d

5.64±0.26

5.68±0.32

5.69±0.37

5.71±0.45

0.0526

TLY, kg

171±5.21c

183±3.61b

191±3.59a

191±4.22a

0.0356

EI, d

6.01±0.07

6.00±0.10

5.99±0.09

5.98±0.08

0.1584

NWL

9.98±0.45

10.01±0.26

10.23±0.15

10.22±0.23

0.0646

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Values with different small letter superscripts in the same column mean a significant difference (p < 0.05). ADG, average daily gain of piglets; ADFI, average daily feed intake of lactating sows; EI, estrus interval; TLY, total lactation yield; NWL, number of piglet weaning litters.

3.2. Semen vaccariae extracts improved the serum hormone levels of lactating sows

There were no significant differences (p > 0.05) in the serum IGF-1, INS, PRL, and E contents on the 107th day of gestation. The serum IGF-1, INS, PRL and E contents were 11.25, 12.50 and 13.13% vs. 9.12, 9.41 and 9.29% vs. 6.35, 12.46 and 12.14% vs. 11.06, 18.36 and 10.93%, respectively, higher (p < 0.05) in the groups treated with the semen vaccariae than those in the control group on the first day of lactation. The serum IGF-1, INS, PRL, and E contents were 7.62, 13.81 and 8.57% vs. 32.86, 43.68 and 50.05% vs. 8.68, 10.95 and 11.03% vs. 9.40, 10.94 and 12.38%, respectively, higher (p < 0.05) in the groups treated with the semen vaccariae than those in the control group, the IGF-1 contents in SV2 were 5.75% vs. 4.82% higher (p < 0.05) than those in SV1and SV3, and the INS contents were increased (p < 0.05) 8.15% vs. 12.94% in SV2 and SV3 compared with SV1 on the 21st day of lactation (Table 4).

Table 4.

Effect of semen vaccariae extracts on the serum hormone levels of lactating sows.

Parameters

Con

SV1

SV2

SV3

p-value

107th day of gestation

IGF-1, ng/mL

136±7.33

139±9.16

138±6.98

137±9.26

0.5672

INS, mIU/L

11.12±0.75

12.30±1.26

11.59±0.96

11.63±1.22

0.6829

PRL, ng/mL

64.02±0.89

65.31±0.95

64.93±0.89

64.86±0.67

0.7596

E, pg/mL

30.06±0.43

31.26±0.59

30.64±0.98

30.43±0.94

0.6524

1st day of lactation

IGF-1, ng/mL

160±9.23c

178±5.62b

180±6.55a

181±8.36a

0.0462

INS, mIU/L

17.65±0.75c

19.26±0.53b

19.31±0.98a

19.29±0.82a

0.0395

PRL, ng/mL

80.26±0.95c

85.36±0.81b

90.26±0.76a

90.00±0.98a

0.0403

E, pg/mL

54.26±0.89c

60.26±0.98b

64.22±0.58a

60.19±0.99b

0.0443

21st day of lactation

IGF-1, ng/mL

210±9.36c

226±5.91b

239±5.24a

228±4.36b

0.0429

INS, mIU/L

21.06±0.95c

27.98±0.86b

30.26±0.64a

31.60±0.68a

0.0356

PRL, ng/mL

70.26±0.95b

76.36±0.65a

77.95±0.87a

78.01±0.68a

0.0356

E, pg/mL

46.27±0.95b

50.62±0.86a

51.33±0.62a

52.00±0.95a

0.0452

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Values with different small letter superscripts in the same column mean a significant difference (p < 0.05). IGF-1, insulin-like growth factor-1; INS, insulin; PRL, prolactin; E, estrogen.

3.3. Semen vaccariae extracts improved the plasma-free amino acid concentration of lactating sows

The plasma Lys, Thr, and Try concentrations were 17.47, 38.18 and 38.70% vs. 10.49, 14.10 and 13.78% vs. 23.27, 54.88 and 24.33%, respectively, higher (p < 0.05) in the groups treated with semen vaccariae than those in the control group, and they were increased (p < 0.05) by 17.63 and 18.08% vs. 3.27 and 2.98% vs. 25.65 and 0.86%, respectively, in SV2 and SV3 compared with SV1 on the first day of lactation. The plasma Lys, Met, Thr, and Try concentrations were 31.56, 56.66 and 56.95% vs. 23.08, 39.40 and 39.02% vs. 12.84, 26.39 and 13.01% vs. 40.50, 90.23 and 42.10%, respectively, higher (p < 0.05) in the groups treated with semen vaccariae than those in the control group on the 21st day of lactation. The plasma Lys and Met concentrations in SV2 and SV3 were increased (p < 0.05) by 19.08 and 19.30% vs. 13.26 and 12.96%, respectively, compared with SV1, and the plasma Thr and Try concentrations in SV2 were 12.01% vs. 11.84 and 35.40% vs. 33.87%, respectively, higher (p < 0.05) than those in SV1 and SV3 on the 21st day of lactation (Table 5).

Table 5.

Effect of semen vaccariae extracts on the plasma-free amino acid concentration of lactating sows.

Parameters

Con

SV1

SV2

SV3

p-value

1st day of lactation

Valine (Val), μg/mL

23.60±0.89

24.63±0.92

24.00±0.86

23.98±0.95

0.5674

Leucine (Leu), μg/mL

15.23±0.63

15.32±0.89

15.33±0.68

15.43±0.98

0.6259

Isoleucine (Ile), μg/mL

7.23±0.56

7.33±0.98

7.35±0.54

7.36±0.76

0.5561

Lysine (Lys), μg/mL

9.56±0.26c

11.23±0.63b

13.21±0.29a

13.26±0.41a

0.0456

Methionine (Met), μg/mL

6.32±0.25

6.36±0.39

6.43±0.98

6.41±0.76

0.3896

Cysteine (Cys), μg/mL

3.65±0.26

3.70±0.62

3.71±0.81

3.70±0.58

0.1254

Phenylalanine (Phe), μg/mL

13.65±0.95

13.70±0.86

13.71±0.65

13.70±0.86

0.3276

Threonine (Thr), μg/mL

31.28±1.31c

34.56±2.35b

35.69±3.24a

35.59±3.69a

0.0472

Glycine (Glu), μg/mL

29.11±1.20

29.23±1.89

30.24±3.12

30.20±3.56

0.0675

Arginine (Arg), μg/mL

21.87±2.21

22.36±3.51

22.52±3.87

21.98±3.21

0.1542

Alanine (Ala), μg/mL

64.25±3.56

65.21±4.25

64.29±5.62

65.22±5.82

0.2681

Serine, Ser, μg/mL

9.71±0.23

9.72±0.62

9.87±0.33

9.85±0.43

0.1583

Glycine (Gly), μg/mL

30.56±3.12

31.26±3.56

31.32±3.81

31.40±3.82

0.2634

Histidine (His), μg/mL

10.26±0.95

10.36±0.98

10.29±0.99

10.39±0.98

0.1587

Tryptophan (Try), μg/mL

5.63±0.98c

6.94±0.89b

8.72±0.86a

7.00±0.69b

0.0421

21st day of lactation

Valine (Val), μg/mL

19.60±0.89

20.36±0.92

20.00±0.75

20.98±0.97

0.4755

Leucine (Leu), μg/mL

13.33±0.67

13.36±0.79

13.37±0.58

13.41±0.72

0.2653

Isoleucine (Ile), μg/mL

5.24±0.66

5.36±0.78

5.37±0.94

5.36±0.97

0.6532

Lysine (Lys), μg/mL

10.36±0.36c

13.63±0.67b

16.23±0.46a

16.26±0.98a

0.0306

Methionine (Met), μg/mL

5.33±0.37c

6.56±0.35b

7.43±0.21a

7.41±0.29a

0.0396

Cysteine (Cys), μg/mL

4.62±0.36

4.71±0.92

4.70±0.93

4.70±0.85

0.2652

Phenylalanine (Phe), μg/mL

10.56±0.55

10.70±0.96

10.70±0.94

10.70±0.99

0.4320

Threonine (Thr), μg/mL

29.82±1.25c

33.65±1.24b

37.69±2.36a

33.70±2.62b

0.0332

Glycine (Glu), μg/mL

26.51±1.23

26.23±1.99

28.22±3.15

28.20±3.96

0.1267

Arginine (Arg), μg/mL

20.89±2.29

21.33±3.49

21.52±3.93

21.42±3.37

0.1643

Alanine (Ala), μg/mL

60.27±3.57

60.25±4.22

60.30±5.21

60.29±5.96

0.2762

Serine (Ser), μg/mL

6.72±0.24

6.72±0.42

6.87±0.63

6.85±0.73

0.2586

Glycine (Gly), μg/mL

37.55±3.62

37.26±3.96

37.32±3.91

37.40±3.89

0.3631

Histidine (His), μg/mL

9.21±0.93

9.31±0.99

9.29±0.89

9.39±0.88

0.1080

Tryptophan (Try), μg/mL

5.63±0.78c

7.91±0.76b

10.71±0.86a

8.00±0.76b

0.0453

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Values with different small letter superscripts in the same column mean a significant difference (p < 0.05).

3.4. Semen vaccariae extracts improved the milk-free amino acid concentration of lactating sows

The milk Lys, Met, Thr, and Try concentrations were 65.86, 117.82 and 66.47% vs. 36.36, 69.70 and 90.91% vs. 13.51, 16.22 and 14.41% vs. 26.79, 44.64 and 48.21%, respectively, higher (p < 0.05) in the groups treated with semen vaccariae than those in the control group; the milk Lys concentration in SV2 was increased (p < 0.05) by 31.33% vs. 30.85%, respectively, compared with SV1 and SV3, the milk Met concentration in SV3 was increased (p < 0.05) by 40.00% vs. 12.50%, respectively, compared with SV1 and SV2, the milk Try concentrations in SV2 and SV3 were increased (p < 0.05) by 14.08% vs. 16.90%, respectively, compared with SV1 on the first day of lactation. The milk Lys, Met, Thr, and Try concentrations were 10.46, 33.39 and 33.48% vs. 51.46, 86.41 and 87.38% vs. 12.14, 24.64 and 33.93% vs. 28.57, 59.52 and 66.67%, respectively, higher (p < 0.05) in the groups treated with semen vaccariae than those in the control group; the milk Lys, Met, and Try concentrations were increased (p < 0.05) by 13.27, 23.08 and 24.07% vs. 13.35, 23.72 and 29.63%, respectively, in SV2 and SV3 compared with SV1, the milk Thr concentration in SV3 was 19.43% vs. 7.45%, respectively, higher (p < 0.05) than that in SV1 and SV2 on the 21st day of lactation (Table 6).

Table 6.

Effect of semen vaccariae extracts on the milk-free amino acid concentration of lactating sows.

Parameters

Con

SV1

SV2

SV3

p-value

1st day of lactation

Valine, Val, μg/mL

1.13±0.08

1.12±0.05

1.13±0.08

1.13±0.06

0.1265

Leucine, Leu, μg/mL

1.52±0.02

1.51±0.03

1.52±0.04

1.52±0.06

0.2634

Isoleucine, Ile, μg/mL

0.89±0.07

0.90±0.09

0.91±0.08

0.90±0.07

0.8237

Lysine, Lys, μg/mL

3.31±0.09c

5.49±0.06b

7.21±0.09a

5.51±0.09b

0.0345

Methionine, Met, μg/mL

0.33±0.02d

0.45±0.04c

0.56±0.02b

0.63±0.03a

0.0202

Cysteine, Cys, μg/mL

2.21±0.07

2.23±0.09

2.21±0.06

2.21±0.08

0.5621

Phenylalanine, Phe, μg/mL

1.68±0.09

1.68±0.06

1.65±0.09

1.68±0.07

0.6892

Threonine, Thr, μg/mL

1.11±0.05b

1.26±0.04a

1.29±0.05a

1.27±0.08a

0.0436

Glycine, Glu, μg/mL

3.82±0.16

3.83±0.26

3.79±0.65

3.82±0.51

0.5281

Arginine, Arg, μg/mL

2.18±0.15

2.16±0.20

2.19±0.19

2.21±0.23

0.6257

Alanine, Ala, μg/mL

3.95±0.30

3.96±0.42

4.00±0.52

4.01±0.32

0.7612

Serine, Ser, μg/mL

3.12±0.33

3.13±0.29

3.10±0.39

3.12±0.42

0.8618

Glycine, Gly, μg/mL

3.86±0.17

3.89±0.26

3.91±0.42

3.90±0.40

0.6213

Histidine, His, μg/mL

0.75±0.01

0.74±0.02

0.75±0.03

0.76±0.03

0.4525

Tryptophan, Try, μg/mL

0.56±0.01c

0.71±0.03b

0.81±0.06a

0.83±0.07a

0.0362

21st day of lactation

Valine, Val, μg/mL

2.01±0.02

2.02±0.02

2.02±0.04

2.04±0.03

0.5628

Leucine, Leu, μg/mL

2.49±0.12

2.50±0.21

2.51±0.11

2.50±0.19

0.6215

Isoleucine, Ile, μg/mL

0.55±0.02

0.52±0.03

0.53±0.04

0.55±0.04

0.4283

Lysine, Lys, μg/mL

11.26±0.22c

13.26±0.34b

15.02±0.35a

15.03±0.62a

0.0473

Methionine, Met, μg/mL

1.03±0.01c

1.56±0.02b

1.92±0.04a

1.93±0.03a

0.0328

Cysteine, Cys, μg/mL

3.94±0.06

3.93±0.07

3.96±0.09

3.97±0.09

0.5621

Phenylalanine, Phe, μg/mL

9.26±0.89

9.27±0.62

9.31±0.98

9.30±0.91

0.6587

Threonine, Thr, μg/mL

11.20±0.12d

12.56±0.32c

13.96±0.53b

15.00±0.92a

0.0437

Glycine, Glu, μg/mL

60.12±1.26

61.02±2.36

61.07±2.61

61.23±3.29

0.0835

Arginine, Arg, μg/mL

10.96±0.12

10.89±0.23

10.92±0.63

1.86±0.52

0.8624

Alanine, Ala, μg/mL

24.45±1.23

24.56±1.96

24.90±2.65

24.89±3.21

0.9561

Serine, Ser, μg/mL

5.46±0.26

5.36±0.62

5.46±0.86

5.64±0.95

0.8624

Glycine, Gly, μg/mL

21.82±3.55

21.86±4.02

21.89±3.92

21.87±4.23

0.9887

Histidine, His, μg/mL

3.76±0.42

3.75±0.46

3.78±0.52

3.80±0.62

0.5612

Tryptophan, Try, μg/mL

0.42±0.01c

0.54±0.03b

0.67±0.05a

0.70±0.03a

0.0406

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Values with different small letter superscripts in the same column mean a significant difference (p < 0.05).

3.5. Semen vaccariae extracts improved the milk composition of lactating sows

The MLR and MPC contents were increased (p < 0.05) by 39.91, 72.77 and 88.26% vs. 23.89, 40.85 and 40.94%, respectively, in the groups treated with semen vaccariae compared with the control group. The MPC contents were 13.69% vs. 13.76%, respectively, higher (p < 0.05) in SV2 and SV3 than those in SV1, the MLR contents in SV3 were 34.56% vs. 8.96%, respectively, higher (p < 0.05) than those in SV1 and SV2, the MFR contents were increased (p < 0.05) by 54.97% vs. 55.53%, respectively, in SV2 and SV3 compared with the control group on the first day of lactation. The MFR and MPC contents were increased (p < 0.05) by 83.41 and 84.13% vs. 30.12 and 35.96% in SV2 and SV3 compared with the control group; the MLR contents were 25.49, 47.06 and 48.04%, respectively, higher (p < 0.05) than those in the control group on the 21st day of lactation (Table 7).

Table 7.

Effect of semen vaccariae extracts on the milk composition of lactating sows.

Parameters

Con

SV1

SV2

SV3

p-value

1st day of lactation

MFR, %

5.33±1.09b

6.87±0.86ab

8.26±0.56a

8.29±0.48a

0.0443

MLR, %

2.13±0.16d

2.98±0.23c

3.68±0.21b

4.01±0.29a

0.0301

MPC, %

11.26±1.23c

13.95±0.86b

15.86±0.59a

15.87±0.64a

0.0298

SNF, %

14.33±1.26

14.62±2.36

14.23±3.25

14.63±3.38

0.5615

21st day of lactation

MFR, %

4.16±1.23b

5.96±1.02ab

7.63±0.96a

7.66±0.84a

0.0326

MLR, %

2.04±0.15c

2.56±0.12b

3.00±0.22a

3.02±0.32a

0.0256

MPC, %

10.26±1.23b

11.32±1.98ab

13.35±0.62a

13.95±1.26a

0.0493

SNF, %

10.27±2.36

10.32±2.31

10.30±3.34

10.29±3.21

0.6235

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Values with different small letter superscripts in the same column mean a significant difference (p < 0.05). MFR, milk fat ratio; MLR, milk lactose ratio; MPC, milk protein content; SNF, solids-not-fat.

3.6. Semen vaccariae extracts improved the milk immunoglobulin composition of lactating sows

The IgM, IgA, and IgG contents were 19.00, 19.18 and 19.10% vs. 64.02, 126.78 and 159.83% vs. 9.38, 19.78 and 22.92%, respectively, higher (p < 0.05) in SV1, SV2, and SV3 than those in the control group, and the IgG contents were increased (p < 0.05) by 9.52% vs. 12.38%, respectively, in SV2 and SV3 compared with SV1, the IgA contents in SV3 were 58.42% vs. 14.58%, respectively, higher (p < 0.05) than those in SV1 and SV2 on the first day of lactation. There were no significant differences (p > 0.05) in the IgM, IgA, and IgG contents among groups on the 21st day of lactation (Table 8).

Table 8.

Effect of semen vaccariae extracts on the milk immunoglobulin of lactating sows.

Parameters

Con

SV1

SV2

SV3

p-value

1st day of lactation

IgM, mg/mL

38.06±3.52b

45.29±2.09a

45.36±2.34a

45.33±2.62a

0.0459

IgA, mg/mL

2.39±0.54d

3.92±0.26c

5.42±0.33b

6.21±0.34a

0.0302

IgG, mg/mL

192±10.21c

210±5.63b

230±12.12a

236±10.36a

0.0289

21st day of lactation

IgM, mg/mL

50.26±3.26

51.33±4.46

51.69±4.68

52.22±4.65

0.5628

IgA, mg/mL

4.01±0.65

4.09±0.98

4.12±0.62

4.23±0.78

0.6258

IgG, mg/mL

240±10.69

239±11.26

241±12.05

242±13.56

0.5495

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Values with different small letter superscripts in the same column mean a significant difference (p < 0.05). IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M.

3.7. Semen vaccariae extracts improved the relative gene expression related to mammogenesis of lactating sows

The relative PRLR, STAT5a, FcRn, CSN2, and LALBA expressions in the groups treated with semen vaccariae were 202, 326 and 327% vs. 192, 262 and 260% vs. 256, 426 and 430% vs. 157, 221 and 225% vs. 212, 215 and 213%, respectively, higher (p < 0.05) than those in the control group on the first day of lactation. The relative PRLR, STAT5a, FcRn, CSN2, and LALBA expressions in the groups treated with semen vaccariae were 102, 226 and 227% vs. 92, 142 and 146% vs. 156, 226 and 230% vs. 147, 147 and 146% vs. 112, 115 and 113%, respectively, higher (p < 0.05) than those in the control group, and the relative FcRn expressions were increased (p < 0.05) by 27.34% vs. 28.91%. Respectively, in SV2 and SV3 compared with SV1 on the 21st day of lactation (Figure 1).

Figure 1.

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The relative gene expression related to mammogenesis on the 1st and 21st day of lactation. The data of relative gene expression in the four Con, SV1, SV2, and SV3 groups, (A) on the first day of lactation, PRLP (Prolactin receptor): 1.00 ± 0.01c, 3.02 ± 0.56b, 4.26 ± 0.35a, 4.27 ± 0.51a, respectively, p = 0.0324; STAT5a (Signal transduction and activator of transcription 5a): 1.00 ± 0.01c, 2.92 ± 0.21b, 3.62 ± 0.16a, 3.60 ± 0.19a, respectively, p = 0.0223; STAT5b (Signal transduction and activator of transcription 5b): 1.00 ± 0.01, 1.01 ± 0.02, 1.02 ± 0.03, 0.99 ± 0.02, respectively, p = 0.0827; (B) on the 21st day of lactation, PRLP (Prolactin receptor): 1.00 ± 0.01c, 2.02 ± 0.21b, 3.26 ± 0.25a, 3.27 ± 0.31a, respectively, p = 0.0434; STAT5a (Signal transduction and activator of transcription 5a): 1.00 ± 0.01c, 1.92 ± 0.11b, 2.42 ± 0.13a, 2.46 ± 0.21a, respectively, p = 0.0334; STAT5b (Signal transduction and activator of transcription 5b): 1.00 ± 0.01, 1.00 ± 0.02, 1.01 ± 0.03, 0.99 ± 0.02, respectively, p = 0.0738; (C) on the first day of lactation, FcRn (Neonatal Fc receptor): 1.00 ± 0.01c, 3.56 ± 0.26b, 5.26 ± 0.34a, 5.30 ± 0.31a, respectively, p = 0.0264; CNS2 (β-Casein): 1.00 ± 0.0c, 2.57 ± 0.21b, 3.21 ± 0.16a, 3.25 ± 0.23a, respectively, p = 0.0261; LALBA (α-Lactalbumin):1.00 ± 0.01b, 3.12 ± 0.23a, 3.15 ± 0.24a, 3.13 ± 0.20a, respectively, p = 0.0359; (D) on the 21st day of lactation, FcRn (Neonatal Fc receptor): 1.00 ± 0.01c, 2.56 ± 0.21b, 3.26 ± 0.31a, 3.30 ± 0.21a, respectively, p = 0.0354; CNS2 (β-Casein): 1.00 ± 0.0b, 2.47 ± 0.22a, 2.47 ± 0.26a, 2.46 ± 0.24a, respectively, p = 0.0376; LALBA (α-Lactalbumin): 1.00 ± 0.01b, 2.12 ± 0.21a, 2.15 ± 0.24a, 2.13 ± 0.20a, respectively, p = 0.0358. Values with different small letter superscripts in the same column mean a significant difference (p < 0.05).

4. Discussion

The lactation period is an essential phase of the reproductive cycle, mainly because the sows have a high demand for nutrients to support milk synthesis. Furthermore, an adequate supply of milk is essential for the growth and survival of piglets (18). Shi et al. added 1% semen vaccariae extracts to mouse feed and found that it stimulated mammary gland development and enhanced lactation potential in mice (19). Wu et al. found that the soy isoflavones improved sows’ lactation performance (20). These conclusions are consistent with the results of this study that the average daily gain of piglets and total lactation yield in SV2 and SV3 were increased compared with the control group. It may be attributed into the fact that the active substances in semen vaccariae stimulate the mammary gland development of sows, increase their milk production and ultimately improve the daily gain of piglets.

In most mammals, prolactin is essential for maintaining lactation, and prolactin suppresses lactation (17). The neuroendocrine system is the central regulatory unit of the mammary gland. Estrogen and prolactin can promote mammary gland development and initiate and maintain animal lactation (21, 22). It has been shown that PRL regulates the lactation process in animal cells through messenger substances cGMP and cAMP, where cGMP promotes milk synthesis, and cAMP accelerates milk secretion (23). Therefore, estrogen can directly promote the proliferation and differentiation of breast cells, promote prolactin secretion, and induce lactation initiation (24). Many different types of cells in the animal body are equipped with receptors that accept insulin-like growth factor, making insulin-like growth factor a good protagonist in the cell signaling process by targeting tissues to promote cell–cell communication or cell division (25, 26). Insulin-like growth factors are closely related to insulin, carrying the same amount of amino acids as insulin and facilitating the anabolic response of growth hormone (27). Cui Nai Ling, traditional veterinary medicine with seme vaccariae as one of the active ingredients, could upregulate prolactin levels, regulate insulin resistance, and improve the glucose and lipid metabolism disorders in rats (28). In the present study, the insulin-like growth factor-1, insulin, prolactin, and estrogen contents were increased in SV2 and SV3 compared with the control group, consistent with the above results. It may be attributed to the fact that the hormone metabolic levels are regulated after sows absorb different degrees of semen vaccariae. Each hormone content is different in various production stages, and the specific mechanism of the effect needs further study.

Fat, lactose, protein, and solids-not-fat are the main components of milk and essential indicators for milk quality evaluation (29). The main carbohydrate in milk is lactose, which is synthetically converted from glucose in the blood (30). The main component of fat is triglycerides, which can be produced in the mammary gland’s epithelial cells or blood (31). Almost 50% of the energy required by newborn piglets comes from the fat in colostrum, so the higher the fat content, the better the survival and health of the piglets (32). In addition, colostrum contains high immunoglobulin concentration, an essential source of the immune ability of piglets (33). In this study, the colostrum fat ratio, colostrum lactose ratio, and colostrum protein content in SV2 and SV3 were increased compared with the control group, indicating that the semen vaccariae improved the growth performance of piglets by improving the milk quality, which was consistent with the results of average daily gain of piglets and total lactation yield of sows above. Lysine, methionine, threonine, and tryptophan are essential amino acids in pig production (34). Dourmad et al. reported that the optimal standardized digestible threonine-to-lysine ratio in feed benefited multiparous gestating sows (35). Samuel et al. found that diets with increased lysine content are needed in late gestation, and increasing the feed allowance in late gestation is necessary to maintain a positive energy balance throughout gestation (36). Bin et al. found that a diet consisting of 0.48% methionine administered during the late gestation period can improve the production performance of sows and maintain their health (37). In this study, the lysine, methionine, threonine, and tryptophan concentration were increased in SV2 and SV3 in plasma and milk on the 1st and 21st day of lactation, indicating that the semen vaccariae promoted the lysine, methionine, threonine, and tryptophan absorption in the feed and its synthesis of these four amino acids, which echoes the results of the protein content in milk above. Kielland et al. showed that increased immunoglobulin G levels in colostrum will improve the levels of immunoglobulin G in piglets and potentially increase the survival of the piglets (38). Quesnel et al. reported that various ingredients that presumably have immuno-modulating effects (such as plant extracts, prebiotics, and probiotics) increased concentrations of IgG, IgA, and/or IgM in sow colostrum when they were provided during the last weeks of gestation (39). In this study, the immunoglobulin A, G, and M contents were increased in SV2 and SV3 compared with the control group on the first day of lactation, indicating that the semen vaccariae greatly affected the early lactation period of sows. In the late lactation period of sows, it may be due to the changes in the physiological metabolism of sows and the decrease in piglets’ demand for milk, so there is no significant difference in immunoglobulin contents in sows, and there is no substantial change in the immunoglobulin contents in sow milk.

The mammary gland must undergo several changes to form lactating glands, such as epithelial maturation and acinar formation processes, which are primarily hormonally regulated (40, 41). Prolactin is a peptide hormone produced by the pituitary gland and other tissue mammary epithelium, which directly affects mammary gland development by binding to the prolactin receptor in myoepithelial cells and indirectly affects mammary gland development by regulating the progesterone release through the pituitary-ovary axis, and prolactin promotes acinar differentiation in combination with progesterone (42). During lactation, prolactin stimulates the JAK2 activation by binding to the prolactin receptor via the JAK–STAT pathway, which phosphorylates STAT5, which subsequently binds to the promoter of the milk protein gene and causes it to be transcribed, resulting in milk production (43–45). In this regard, STAT5a can synergize with the glucocorticoid receptor to induce milk protein gene expression, and progesterone and glucocorticoids can inhibit apoptosis in MECs (46–48). The JAK–STAT pathway also induces the members of the SOCS protein family expression, which negatively regulates the prolactin regulatory pathway, thereby co-regulating mammary gland lactation (49). Suckling of milk by young animals also stimulates prolactin release, which induces myoepithelial cell contraction, allowing milk to pass from the acinar into the ducts (50). The FcRn gene plays a vital role in the immunoglobulin G transport during colostrum formation and has a secretory role in epithelial cells (51). CSN2 gene is a member of the beta-casein family; there are two types of casein protein, beta (encoded by this gene) and kappa, which are secreted in animal milk. Beta casein is the principal protein in animal milk and the primary source of essential amino acids for suckling young animals (45, 52). The LALBA gene encodes alpha-lactalbumin, a principal protein of milk (53). Alpha-lactalbumin forms the regulatory subunit of the lactose synthase heterodimer, and beta 1,4-galactosyltransferase forms the catalytic component. These proteins enable lactose synthase to produce lactose by transferring galactose moieties to glucose (54). In this study, the relative PRLR, STAT5a, FcRn, CSN2, and LALBA expressions in the groups treated with semen vaccariae were higher than those in the control group. The results of the relative PRLR, STAT5a, and STAT5b expressions were consistent with the PRL contents in serum on the 1st and 21st day of lactation, indicating that the semen vaccariae can improve the mammogenesis by regulating the gene expressions in the JAK–STAT signaling pathway. The relative FcRn expression results were consistent with the immunoglobulin G contents in colostrum, and the milk protein contents on the 1st and 21st day of lactation were compatible with the relative CSN2 and LALBA expressions, indicating the semen vaccariae can improve the milk quality by regulating the CSN2 and LALBA expression.

5. Conclusion

In this study, the optimum dosage was 3.0 g/kg semen vaccariae, which increased the average daily gain of piglets and total lactation yield during the 11–21 days and 1–21 days of lactation, increased the serum insulin-like growth factor-1, insulin, prolactin, and estrogen contents on the 1st and 21st day of lactation, increased the plasma and milk Lysine, Methionine, Threonine, and Tryptophan concentrations on 21st day of lactation, increased the milk fat ratio, milk protein content, relative PRLR, STAT5a, FcRn, CSN2, and LALBA expressions on the 1st and 21st day of lactation.