Dietary Semen Vaccariae enhances mammary development and lactation potential in rats

Abstract

An experiment was conducted to study the effects of dietary Semen Vaccariae (SV) on mammary development and lactation potential in rats. Forty-eight Sprague–Dawley female rats after parturition were selected and assigned to two groups randomly, with 24 rats each. Control rats were offered basal diets from the beginning to the end of the lactation period. A total of 1% SV was added to the basal diet to form an experimental diet. After parturition, the litter size was culled to eight pups per dam. Estimated milk yield was improved 29.82% (P<0.05) by feeding SV on day 18 of lactation. Serum growth hormone, insulin-like growth factor-1 and prolactin concentrations in dams were significantly higher (P<0.05) in the SV group than in the control group on day 18 of lactation. Dietary SV increased wet weight and DNA contents of dam mammary (P<0.05). The results suggest that dietary SV stimulates mammary development and promotes lactation in rats.

Keywords:

• Semen Vaccariae
• mammary
• lactation
• endocrine
• rats

Introduction

Currently absence or deficiency of milk yield among mammalian species during the postpartum period is a frequent question (Amir and Cwikel 2005; Papadopoulos et al. 2008). Many traditional Chinese herbs have been used to improve lactation potential, with natural raw materials, less side effects and no tolerance and drug-resistance. The most common of these is Semen Vaccariae (SV), a seed of Vaccaria segetalis (Nack) Garcke, which has been shown to be the most effective at increasing milk production (Shi and Shan 2007). SV has also been used as a Chinese drug for invigorating circulation, regulating menstrual dispel edema. The vaccaria is an annual plant that grows in China and parts of central and southern Europe (Meesapyodsuk et al. 2007). It is also known as the cowherb or cow soapwort. Currently herbal feed additive products for improving lactation potential mainly resulted from folkloric or traditional prescription, However, which have the disadvantage of instable effect. Unfortunately, there is no scientific research on how the herbs actually work and which ones are valid. The objective of the study was to investigate the effects of dietary SV on mammary development and lactation potential in rats.

Materials and methods

Sprague–Dawley female rats at approximately 3 months old were mated on the same day. They were housed individually in metal wire mesh-bottomed cages and isolated in a controlled environment of 25°C and 50% relative humidity with a 12-h light:dark cycle and free access to water. After parturition, the litter size was culled to eight pups per dam. The day on which a litter was found was designated as day 1 of lactation. Forty-eight Dam rats were selected and assigned to two groups randomly, with 24 rats each. Control rats were offered basal diets (Table 1) ad libitum from the beginning to the end of the lactation period. A total of 1% SV was added to the basal diet to form experimental diet. Sixteen dams from each group were randomly selected to determine lactation performance. Dam weight, litter weight and average pup weight were recorded on days 1 and 22 of lactation. Cumulative feed intake was recorded during the lactation period and average daily intake was estimated, also. In addition, the remaining eight dam rats from each group were used to determine the influence of dietary SV on dam serum hormone and mammary development.

Table 1.  Ingredients and chemical composition of basal diets for female rats.

Ingredients	Percentage	Composition^a	Percentage
Corn	29.71	Crude protein	19.88
Soybean meal	20	Gross energy (MJ/kg)	17.1
Flour	16	Methionine	0.36
Wheat bran	12	Lysine	0.95
Sorghum	12	Calcium	0.92
Fish meal	5	Phosphorus	0.76
Bone meal	2
Brewers dried yeast	1
Cod-liver oil	1
Salt	1
Mineral premix^b	0.25
Vitamin premix^c	0.04
Total	100
^aCalculated analysis data.
^bConsist of ingredients (%): MnSO4 · H2O, 6.65; ZnSO4 · H2O, 3.39; CuSO4 · 5H2O, 0.97; FeSO4 · 7H2O, 6.97; KI (Including I, 1%), 2.2; Cornstarch, 79.82.
^cConsist of ingredients (kg^−1): VA, 4000 IU; VD3, 1000 IU; VE, 50 IU; VK3, 50 µg; VB1, 6 mg; VB2, 6 mg; VB6, 7 mg; niacin, 30 mg; pantothenic acid, 16 mg; folic acid, 2 mg; pyridoxine, 0.2 mg.

Daily milk yield on day 18 of lactation was determined by a stepwise forward multiple regression equation (Sampson and Jahansen 1984) as follows:

where yield=total milk yield per litter (g); weight=average pup weight (g); gain=average rate of pup weight gain (g/day) and 8=litter size.

Eight rats per group were killed by CO₂ gas for mammary tissue collection on day 11 of lactation. Mammary gland tissues were excised, weighed, snap frozen in liquid nitrogen and stored at −80°C. DNA from frozen mammary tissue was extracted using MagExtractor-Genome (Toyobo, Osaka, Japan) according to the manufacturer's instructions. The DNA concentration was determined by absorbance readings in a Pharmacia Biotech Ultrospec 3000. Total mammary RNA was extracted from frozen mammary tissue using Trizol® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Blood was collected by cardiac puncture, centrifuged (1500×g for 10 min) and the serum stored at −80°C until analysis. Serum growth hormone (GH), prolactin (PRL), 3,3′,5-Triiodothyronine (T₃) and thyroxine (T₄) were measured by using commercial ¹²⁵I-labeled radioimmunoassay (RIA) kits (BNIBT, Beijing, China), respectively. Insulin-like growth factor-1 (IGF-1) was also measured by using commercial ¹²⁵I-labeled RIA kit (Biocode-Hycel, Liege, Belgium).

The analysis of variance (ANOVA) for all these data was used in the software of SAS 9.0 (SAS Institute Inc., Cary, NC, USA). Individual differences between means were tested by Ducan's Multiple Range Test.

Results and discussion

The estimated milk yields of the SV group were 29.82% (P<0.05) higher on day 18 of lactation than these of control group (Table 2). The weight and feed intake did not differ between control and SV group. It shows that SV may put more food nutrients into milk. The pup rats that drink more milk are more apt to become a heavier weight. During days 1–21 of lactation, compared with control group, weight gain of pup rats in the SV group was improved by 22.69% (P<0.05).

Table 2.  Effect of dietary SV on milk yield, feed intake and weight of dam rat and weight gain of pup rat.

Treatments	Control	SV
Milk yield (g/day/dam)	21.73±0.65^b	28.21±0.97^a
Maternal weight (g)	268.83±3.65^a	265.25±5.09^a
Daily feed intake (g/day/dam)	44.96±0.80^a	46.80±0.96^a
Pup rat weight (g)	30.57±0.41^b	36.23±0.51^a
Daily weight gain of pup rat (g/day)	1.19±0.02^b	1.46±0.02^a
Note: Values are means±SD, n=16. ^abMeans in a row without a common letter differ, (P <0.05).

It is well established that many hormones, including GH, IGF-1 and PRL, are necessary for the growth and differentiation of the mammary gland (Camarillo et al. 2001; Naylor et al. 2003; Kleinberg and Ruan 2008). In the present study, it was found that dietary SV enhances lactation potential possibly due to improving serum GH, IGF-1 and PRL concentrations in dams (Table 3).

Table 3.  Effect of dietary SV on serum hormone concentrations in dam rats.

Hormone	Control	SV
GH (mg/ml)	19.32±0.49^b	21.67±0.57^a
IGF-1 (ng/ml)	104.76±3.02^b	128.06±3.57^a
PRL (ng/ml)	38.89±1.84^b	46.77±2.09^a
T3 (ng/ml)	0.95±0.02^a	0.93±0.03^a
T4 (ng/ml)	81.97±5.63^a	87.97±6.27^a
Note: Values are means±SD, n=8. ^abMeans in a row without a common letter differ, (P <0.05).

Milk yield is a function of the number and activity of secretory cells present in the mammary gland. Changes in the cell number during lactation may be responsible for variations in milk yield (Capuco et al. 2001; Boutinaud et al. 2004). Total tissue DNA content may be used as an accurate indicator of cell number and, therefore, development (Quarrie et al. 1996). Since mammary RNA is intimately related to the biosynthesis of protein, this nucleic acid may be used as an indication of the cell's synthetic potential (Winick and Noble 1965). Dams offered diet SV for the lactation period showed a significant increase (P<0.05) in total mammary DNA, but the RNA content per cell did not change (Table 4). And as a result the RNA:DNA ratio was significantly reduced (P<0.05). This may suggest that the herb may promote the proliferation of mammary epithelial cells.

Table 4.  Effect of dietary SV on wet weight and DNA and RNA levels of dam rat mammary.

	Control	SV
Mammary weight, wet (g)	12.33±0.48^b	14.03±0.40^a
Mammary DNA (mg/g)	1.62±0.06^b	1.85±0.07^a
Mammary RNA (mg/g)	11.26±0.43^a	10.49±0.47^a
Mammary RNA:DNA	6.95±0.42^a	5.67±0.37^b
Note: Values are means±SD, n=8. ^abMeans in a row without a common letter differ, (P<0.05).

In summary, it can be concluded that lactating rats offered a diet with herb can induct hormone levels, and thus promoting mammary development, which resulted in rich lactational performance. This increase in lactational ability was as a result of rapid secretory cell proliferation and a marked increase in cellular activity. Future investigations of herbs in lactation are needed, and not just to clarify safety issues, although these concerns are paramount. It is reasonable to expect that systemic interdisciplinary study of plants with lactation-modulating reputations would reveal new therapies and provide research tools for a deeper understanding of human lactation. Further studies are certainly warranted to elucidate valid components and dosage of the herb and its effects on the lactating breast.

Acknowledgements

The work was supported by Doctoral Fund of Ministry of Education of China (20092325120008), the earmarked fund for China Agriculture Research System (CARS-36) and Key Research Program of Heilongjiang Province (GA07B201).