- Email: [email protected]
The oral administration of thermophile-fermented compost extract and its influence on stillbirths and growth rate of pre-weaning piglets
Research in Veterinary Science 93 (2012) 137–142
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
The oral administration of thermophile-fermented compost extract and its influence on stillbirths and growth rate of pre-weaning piglets Hirokuni Miyamoto a,b,⇑, Hiroaki Kodama b, Motoaki Udagawa c, Kenichi Mori a,b, Jiro Matsumoto d, Hatsumi Oosaki e, Tatsuo Oosaki e, Masayuki Ishizeki f, Daisuke Ishizeki f, Ryusuke Tanaka g, Teruo Matsushita g, Yuriko Kurihara h, Hisashi Miyamoto i a
Japan Eco-science Co. Ltd. (Nikkan Kagaku), 11-1-2F Shiomigaoka-chou, Chuou-ku, Chiba 260-0034, Japan Graduate School of Horticulture, Chiba University, 648 Matsudo, Chiba 271-8501, Japan Keiyo Plant Engineering Co. Ltd., 2-8-8 Ichikawaminami, Ichikawa-city, Chiba 272-0033, Japan d Keiyo Gas Co. Ltd., 2-8-8 Ichikawaminami, Ichikawa-city, Chiba 272-0033, Japan e Oosaki Swine Business Ltd., 1195-1 Takikubo-chou, Maebashi, Gunma 371-0235, Japan f Ishizeki Swine Farm Ltd., 1030 Komeno, Fujimimura, Seta, Gunma 371-0111, Japan g Department of Food Science and Technology, National Fisheries University, 2-7-1 Nagatahonchou, Shimonoseki, Yamaguchi 759-6595, Japan h Graduate School of Health Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyou-ku, Tokyo 113-8510, Japan i Miroku Co. Ltd., 706-27 Iwaya, Kitsuki, Oita 873-0021, Japan b c
a r t i c l e
i n f o
Article history: Received 16 October 2009 Accepted 20 June 2011
Keywords: Thermophile Compost Pig Foetal death Development
a b s t r a c t Food produced via fermentation with mesophilic bacteria has been used to confer health benefits. In contrast, mammalian physiological responses to the intake of thermophile-fermented products have not been thoroughly investigated. We examined the effects of administering a compost extract consisting of fermented marine animals with thermophiles, including Bacillaceae, to pregnant sows and piglets. Retrospective studies were performed on two different swine farms (n = 330–1050 sows). The rate of stillbirth was markedly lower in all parities of the compost extract-fed group compared to those of the control group (p 5 0.001). Additionally, the birth to weaning period of newborns was significantly shorter (p < 0.0001), while the ratio of weanlings per liveborn piglets was increased by more than 6.5% in the compost extract-fed group. Thus thermophiles and their products in the compost extract might promote growth and reduce stillbirths of piglets during the birth to weaning period. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The mammalian gastrointestinal tract is a complex ecological environment that is colonized with 100 trillion (1014) microorganisms (Ley et al., 2006), and dominated by members of the Bacteroidetes, Firmicutes, and Archaea (Bäckhed et al., 2005; Eckburg et al., 2005; Ley et al., 2006). Due to the diversity and complexity of these microorganisms, the host-microbe relationship has been poorly studied. Probiotics are live microorganisms, which, when ingested in sufficient amount, confer a health benefit to the host (Fuller, 1991; Kyriakis et al., 1999; Broom et al., 2006). Fermented foods and their included microbes have been commercialized as healthy food or feed and have multiple physiological effects on the health
Abbreviations: FAA, free amino acid; TDN, total digestible nutrients.
⇑ Corresponding author. Address: Japan Eco-science Co. Ltd., 11-1-2F Shiomigaoka-chou, Chuou-ku, Chiba-city, Chiba 260-0034, Japan. Tel.: +81 43 302 2322; fax: +81 43 302 2123. E-mail address: [email protected] (H. Miyamoto). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.06.018
of humans and livestock animals (Broom et al., 2006; Hong et al., 2008; Chen et al., 2009). Most probiotic microbes in fermented foods or feeds are mesophilic microbes. At present, it is not understood whether other microbes, e.g., extremophiles that contain Archaea and their metabolites, have probiotic effects on mammalian health. Recently, we reported the microbial diversity in a unique compost used as a fertilizer in Japan (Niisawa et al., 2008). This compost is a fermented marine animal resources by-product and includes small fish, crabs, and shrimps. These substrates are rapidly fermented at high temperature (approximately 75 °C) by fermentation-associated self-heating, and the final product contains several extremophiles, including thermophiles (Niisawa et al., 2008). The level of Firmicutes decreases during the maturation phase of fermentation in most other composts (Ishii and Takii, 2003; Takaku et al., 2006), but Firmicutes (mainly Bacillaceae) remain dominant in this compost. Strains with 16S rDNA sequences similar to those of Bacillus thermoamylovorans and Bacillus thermocloacae are the predominant bacteria in this compost (Niisawa et al., 2008). In addition, the compost contains a characteristic
138
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
bacterium that produces a cyclic lipopeptide with antifungal activity (Niisawa et al., 2008). Another research group has also reported the isolation of two or more bacteria that produce thermostable exo- and endo-chitinases in this compost (Sakai et al., 1994, 1998). Several Japanese farmers use this compost as an organic fertilizer. The compost is distributed over the soil, or its extract is sprayed onto the leaves of plants. Some of these plants have been used as a feed for chickens and pigs, and composting thermophilic bacteria on the plant surface are ingested by these animals. The thermophilic bacteria in the compost might affect the health of these animals in a direct and/or indirect manner. We initiated an investigation into the effects of the oral administration of an extract of this compost on several animal species. The addition of faeces from compost-fed pigs into the compost process of pig faeces increased the temperature of the compost by 10 °C over that achieved when faeces from noncompost-fed pigs was added. Fermentation at this higher temperature (60–90 °C) reduced the unpleasant odor and shortened the fermentation period. Thus, many Japanese stockbreeders for chickens or pigs now use the compost extract as a feed additive. In addition, this compost or its extract has been used as a feed additive for flatfish Paralichthys olivaceus in a farm and was reported to improve the health of the fish and raise the level of free amino acids in muscle tissue (Tanaka et al., 2010). In this paper, we identify additional effects of this compost extract based on observations of long-term administration to pigs, including prevention of stillbirth in sows and growth promotion in their piglets. To our knowledge, the study presented here is the first to investigate mammalian physiological responses to the oral administration of thermophile-fermented compost extract.
2. Materials and methods 2.1. Animals Sows (crossbred Landrace Large White) and their piglets (crossbred sows Duroc) were conventionally maintained at two Japanese farms (A and B). All pigs were fed a commercial feed (Table 1) and potable water ad libitum. Each sow and her piglets were housed in an individual pen (2.5 m 1.0 m) during the birth to weaning period. The housing area was maintained with 65–80% humidity and ventilation 0.6 m3/min. The temperature of the area was at 18–35 °C dependent on the seasonal changes. The individual piglets in each pen were selected at random and their daily body weights obtained. When the body weight of piglets reached 6.0–6.5 kg, the piglets were weaned and then housed together in another pen (2.5 m 1.8 m) (30 piglets per pen). All treatments of pigs on Farm A and Farm B (Gunma, Japan) were conducted in accordance with the institutional animal care guidelines for Oosaki Swine Business Ltd., Japan, and the Ishizeki Swine Farm Ltd., Japan, respectively. Sow management was according to individual farmspecific guidelines in order to most efficiently produce piglets (Leenhouwers et al., 2003). 2.2. Feeding conditions As seen in Table 1, sows were given two types (X and Y) of sowspecific commercial feed (Ryoumou Bussan Co. Ltd., Japan). The sows were usually given a restricted volume of feed Y (3–4 kg per day). From seven days before birth until all piglets were
Table 1 Composition of the diets in the farms. Sow feeda
Item
a
Piglet feedb
X
Y
I
II
III
IV
Component (%) Protein Lipid Fiber Ash Calcium Phosphate TDNc
17.00 4.00 4.00 6.50 0.85 0.65 77.00
12.00 2.50 4.50 6.00 0.80 0.65 74.00
18.50 5.00 3.00 8.50 0.65 0.55 83.50
18.50 4.00 3.00 8.50 0.65 0.55 80.00
20.50 4.50 3.00 6.50 0.70 0.06 79.00
13.50 4.00 4.00 5.00 0.55 0.40 78.00
Materials (%) Corn Soybeans Fish meal powder Rice bran Rice–lipid bran premixd Rice–wheat bran premixe Animal fat & feed additive premixf Non-animal fat & feed additive premixg Animal fat & lactose & feed additive premix Ah Animal fat & lactose & feed additive premix Bi Lactose & feed additive premixj Whey & fish meal premixk
63.27 27.00 2.00 0.00 0.00 2.03 5.70 0.00 0.00 0.00 0.00 0.00
76.32 15.50 0.00 0.00 0.00 0.93 0.00 8.15 8.15 0.00 0.00 0.00
44.00 21.00 0.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 5.71 18.00
54.00 24.00 0.00 1.00 0.00 0.00 0.00 0.00 11.00 0.00 0.00 10.00
59.77 32.50 2.00 0.00 0.02 0.00 0.00 0.00 0.00 5.71 0.00 0.00
74.38 21.00 0.00 0.00 0.07 0.00 4.55 0.00 0.00 0.00 0.00 0.00
Sows were given the sow feed X or sow feed Y (see Materials and methods). Piglets were given the piglet feeds, I, II, III, IV as described in Materials and methods. TDN shows total digestible nutrient. d Rice–lipid bran premix contains a mixture of rice bran and lipid bran. e Rice–wheat bran premix contains a mixture of rice bran and wheat bran. f Animal fat & feed additive premix contains a mixture of animal fat, calcium phosphate, calcium carbonate, salt, yucca foam extract, alfalfa meal, Bacillus subtilis, Saccharomyces cerevisiae, and lactic acid bacteria. g Non-animal fat & feed additive premix contains calcium phosphate, alfalfa meal, yucca foam extract, calcium carbonate, salt, B. subtilis, S. cerevisiae, and lactic acid bacteria. h The premix A contains a mixture of animal fat, lactose, calcium phosphate, calcium carbonate, yucca foam extract, citrate, lactate, salt, B. subtilis, S. cerevisiae, and lactic acid bacteria. i The premix B contains a mixture excluded S. cerevisiae from the premix A. j Lactose & feed additive premix contains a mixture of lactose, plant fat, calcium phosphate, calcium carbonate, yucca foam extract, citrate, lactate, salt, B. subtilis, S. cerevisiae, and lactic acid bacteria. k Whey & fish meal premix contains a mixture of whey and fish meal powder. b
c
139
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
weaned, sows were given 5–6 kg per day of feed X instead of feed Y. Piglets were given four types of commercial feed (I–IV, see Table 1). From three to 28 days after birth (during the birth to weaning period and about 5–10 days after weaning), piglets were provided feed I (Nisshin-Marubeni Co. Ltd., Japan). After weaning, the piglets (from 28 to 34 days after birth) were fed with feed II (Nisshin-Marubeni Co. Ltd., Japan). From 35 days after birth until the body weight of the piglets exceeded 30 kg, they were fed feed III (Ryoumou Bussan Co. Ltd., Japan). Thereafter, piglets (body weight between 30 kg and 70 kg) were provided feed IV (Ryoumou Bussan Co. Ltd., Japan). 2.3. Treatment of compost The compost used in this study was made from marine animals and coffee residues by a repeated fed-batch fermentation system with three bioreactors, as described in our recent paper (Niisawa et al., 2008). Bacteria in this compost were mainly Bacillaceae, and the 16S rDNA sequences of major bacterial species showed high similarity to those of B. thermoamylovorans and B. thermocloacae. This compost has been marketed as an organic fertilizer or a fermentation feed for pigs and chickens (Miroku Co. Ltd., and Keiyo Plant Engineering Co. Ltd., Japan). The compost was diluted to 1/ 100 with potable water (as v/v), and the resulting suspension incubated aerobically at 60 °C for 10–24 h. The compost suspension was then filtered through a nylon mesh (pore size 100 lm). This compost extract (pH 7.5–8.5) contained <99.9% H2O, <0.1% protein, <0.1% lipid, <0.1% fiber, <0.1% ash, <50 ppm lactate, and <50 ppm butyrate, indicating that the compost extract itself included negligible nutrients. The number of bacteria in the compost extract was assessed as <5.0 106 CFU/ml thermophiles, after culture on nutrient agar (1% beef extract, 1% peptone, and 0.5% NaCl) at 55 °C. The administered aqueous solution of 0.4% (v/v) compost extract was supplied as drinking water to the pigs. After dilution, the water contained <0.2 ppm butyrate and lactate. On Farm A, all pigs housed from February 2004 to the end of 2007 were fed the extract. When administration of the compost extract began in February 2004, the concentration was gradually increased from 0.2% to 0.4% (0.1% increment per week). During the three-year period, the supply of the compost extract was interrupted for one month in October 2004, due to maintenance of the water supply system. All pigs on Farm A were housed from January 2001 to January 2004 and supplied compost extract-free potable water. On Farm B, all pigs housed from July 2006 to June 2008 were provided an aqueous water solution containing 0.4% (v/v) compost extract. Group B pigs, housed from January 2004 to June 2006 were provided compost extract-free potable water. Farms A and B are in the same area of Gunma Prefecture (10 km distance between these two Farms). While the pigs in Farm A were supplied with the compost extracts (2004–2007), the number of stillbirths under the compost extract-free condition was investigated at Farm B (2004–2006). Thus, the effects of climatic conditions between 2004–2006 on stillbirths would be insignificant. 2.4. Data analyses In order to determine which non-parametric or parametric statistical analyses are suitable to all subjects, P values were calculated by using the Kolmogorov–Smirnov test. When the P values were less than 0.05, a non-parametric statistic analysis was suitable for comparisons between two subjects. Here all the data were statistically analyzed using the Mann–Whitney U test (Wilcoxon signed-rank test) (one category in the control group vs. the same category in the compost extract group housed on the same farm). All data were hypothesized as individual subjects, since the body condition of sows varied. Statistical analyses were carried out with
StatView-J 4.02 software for Macintosh. All data are expressed as mean ± SE. 3. Results 3.1. Effects of the thermophile compost on foetal death of piglets The effects of thermophile compost as a feed additive for pigs on foetal deaths were investigated (Table 2). On Farm A, the number of newborn piglets per sow did not differ between the sows administered potable water (control group) and those administered compost solution (compost group). The number of liveborn piglets per sow tended to be higher in the compost-group than the control group (p = 0.082; vs. control-group). A significant difference was obtained for the number of stillbirths (p < 0.0001; vs. control group) and for the rate of stillbirths (p < 0.0001). The number of stillbirths per delivery on Farm B was also significantly reduced (p = 0.0006; vs. control group) (Table 2), showing a significant reduction in the rate of stillbirths in the compost group (p = 0.001; vs. control group). In Farm B, the total number of newborn piglets per sow slightly decreased (p = 0.0039; vs. control group), although the number of liveborn piglets in the compost group was mostly unchanged from that of the control group. Thus, these data suggest that the oral administration of the compost solution to sows might reduce stillbirths. 3.2. Seasonal changes in the rate of stillbirth We detected whether seasonal changes in the stillbirth rate were affected by administration of the compost solution. As seen in Fig. 1a and b, 12–15 sows farrowed, and 150–170 piglets were born every month on Farm A. The stillbirth rate was high in the winter and gradually decreased in the spring in the control group. These seasonal changes in the rate of stillbirths were also evident in the compost group. The lowest rate of stillbirths was seen in early spring (March–April). Each monthly rate of stillbirths between the control and compost groups tended to be different (p < 0.1), except for the period from November to December. Similar results were observed on Farm B (data not shown). These
Table 2 Stillbirth rates for two different farms. Category
Control group
Compost group
Farm A Total number of sows Total number of newborns Total number of stillbirths Newborn piglets per sow Liveborn piglets per sow Stillbirth per sow Rate of stillbirth (%)
986 11,568 1386 11.74 ± 0.10 10.34 ± 0.09 1.41 ± 0.06 11.74 ± 0.10
1050 12,171 1055 11.59 ± 0.11 10.58 ± 0.11a 1.01 ± 0.05b 8.60 ± 0.40b
Farm B Total number of sows Total number of newborns Total number of stillbirths Newborn piglets per sow Liveborn piglets per sow Stillbirth per sow Rate of stillbirth (%)
330 3906 596 11.84 ± 0.18 10.34 ± 0.09 1.81 ± 0.11 15.41 ± 1.05
421 4707 565 11.18 ± 0.15c 9.84 ± 0.14 1.34 ± 0.08d 11.42 ± 0.69e
In Farm A, births were recorded from January 2001 until January 2004, (control group) and from February 2004 until February 2007 (compost group). In Farm B, births were recorded from July 2004 until June 2006 (control group) and from July 2006 until June 2008 (compost group). All data are shown as the mean ± SE. a p < 0.1. b p < 0.0001. c p < 0.005. d p < 0.001. e p < 0.01 (vs. control group).
140
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
iod (three years). The control group showed a lower value for the average of parity than the compost group (3.55 vs. 4.15 in Farm A; 3.39 vs. 3.98 in Farm B). Although the sows of the control group have a potential for showing a lower stillbirth rate than the sows of the compost group with respect to the number of pregnancies, the results showed a marked reduction in the stillbirth rate of the compost group. 3.4. Effects of the thermophile compost on the growth of piglets during birth to weaning The effects of administration of the compost extract to sows on the growth of their offspring during the lactation period were investigated on Farm A (n = 9290–10,403) (Table 4). The average number of weanlings per sow was increased by about 4.7% in the compost group compared to that of the control group (p < 0.0001). In addition, the ratio of weanlings to liveborn piglets also increased by about 6.5% in the compost group (p < 0.0001). Therefore, the oral administration of the compost solution to the lactating sow apparently increased the health of the piglets during birth to weaning. The body weights of the piglets just after weaning were similar in the two groups. It is worthwhile to note that the lactation period was shortened by about three days in the compost group in comparison to the control group (p < 0.0001). The seasonal trend in weaning duration is shown in Fig. 2. The lactation period was shorter in the summer than in the winter, as seen in the control group. The lactation period of the compost group in the winter was nearly the same as that of the control group in the summer, indicating that the administration of the compost extract to the sow enhanced the growth of their piglets during the lactation period. This duration was significantly shortened (p < 0.0001), especially in January and February (p < 0.05), May and June (p < 0.05), July and August (p < 0.05), and November and December (p < 0.0001). Thus, administration of the compost extract to sows enhanced the growth of their piglets until weaning. 4. Discussion and conclusions
Fig. 1. Seasonal changes in number of sows (a), number of newborns (b), and rate of stillbirth (c) in Farm A. The year was divided into six periods of two months each. The data were obtained by counting pig numbers for three years. Since the counting of newborns, their sows, and stillbirth rate was carried out every month, averages were calculated for a two-month unit (n = 6 per period, except January–February (n = 7)). The error bars show mean ± SE. Open circles indicate the data in the control group, while closed circles indicate the data in the compost extract-fed group. ⁄ p < 0.05, and ⁄⁄p < 0.01 (vs. control group).
observations indicate that the rate of stillbirths of the sows of the compost group was stable at a low level during all seasons. 3.3. Effects of sow pregnancy number on stillbirth rates of their piglets We investigated the relationship between parity and stillbirth rate on Farms A and B. The stillbirth rate increased according to the increase in parities in both groups. However, the stillbirth rate in the compost group was markedly lower for all parities when compared with that of the control group (Table 3). The stillbirth rate significantly increased in the control sows of parities 7 and greater, but the increase in stillbirth rate was significantly lower in the compost group. These observations suggest that the increase in stillbirth rate according to the increase in parities tended to be alleviated by administration of the compost extract to sows. This result indicates the importance of parity. We determined the average of parity in both groups during the experimental per-
In this paper, the physiological effects of the oral administration of the extract of thermophile-fermented compost to pigs are addressed in regard to the stillbirth rate of newborns and growth of the piglet. The retrospective studies indicated reduction in the stillbirth rate at two different swine farms where the compost solution was administered. The stillbirth rate in the compost group was markedly lower in all parities of sows compared to the control group. The shorter lactation period in the compost group indicates growth promotion of piglets in addition to the increased number of weaned piglets. Taken together, the preservation and/or improvement of the healthy condition of the sows are important for normal growth of fetuses and newborns (Foxcroft, 1997), and the intake of thermophile-fermented compost extract is apparently beneficial for sustainment of the healthy condition of the host. Possible risk factors for stillbirth have been surveyed (Zaleski and Hacker, 1993; Andersen et al., 2002; Leenhouwers et al., 2003; Smith and Fretts, 2007), but factors that reduce the stillbirth rate have rarely been reported. Recently it has been reported that a microbial feed, which contain mesophilic Bacillus species, reduced mortality of pigs during the growing-finishing period. (Davis et al., 2008). The approximately 570-bp-long 16S rDNA sequences that had been amplified from total DNA of the bacteria in the compost were determined, and the results indicated that the spore-forming bacteria such Bacillus or Bacillus-related genera were predominant in the compost (Niisawa et al., 2008). Most of these bacteria form endospores, and thus pigs might ingest spores in addition to vegetative forms. Compost is preserved in a desiccated condition until
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
141
Table 3 Relationship between parities and stillbirth rates for Farm B. Parities
Control group
Compost group
Number of sows
Stillbirth rate
Number of sows
Stillbirth rate
Farm A 1–3 4–6 7 and greater
416 248 101
12.00 ± 0.51 13.61 ± 0.62 16.57 ± 1.12a,b
269 203 134
8.52 ± 0.59c 10.14 ± 0.69d,e 12.33 ± 0.88f,g
Farm B 1–3 4–6 7 and greater
171 172 67
11.74 ± 1.02 15.13 ± 1.19 27.85 ± 3.29b,h
143 149 112
9.01 ± 1.24i 11.35 ± 1.03j 14.92 ± 1.43k,l
The parities express the number of times the sow had been pregnant. All data are shown as the mean ± SE. a p < 0.001 vs. parities 1–3 for the control group. b p < 0.01 vs. parities 4–6 within the same group. c p < 0.0001 vs. parities 1–3 in the control group. d p < 0.0001 vs. parities 4–6 within the compost group. e p < 0.001 vs. parities 1–3 in the same group. f p < 0.001 vs. parities 7 and greater in the control group. g p < 0.0001 vs. parities 1–3 in the same group. h p < 0.0001 vs. parities 1–3 in the control group. i p < 0.1 vs. parities 1–3 in the control group. j p < 0.05 vs. parities 4–6 in the control group. k p < 0.01 vs. parities 1–3 within the compost group. l p < 0.001 vs. parities 7 and greater in the control group.
Table 4 Ratio of weanlings and the lactation period. Category
Control group
Compost group
Total number of weanlings Number of weanlings per sow Weanlings per liveborn piglets (%) Duration of the lactation period (days)
9290 9.44 ± 0.08 87.02 ± 1.52 24.63 ± 0.35
10,403 9.88 ± 0.08a 93.56 ± 0.51a 21.84 ± 0.27a
These data were obtained for Farm A (2001–2007). The numbers corresponding to total newborns (including living and dead) and liveborn piglets are shown in Table 1. All data are shown as the mean ± SE. a p < 0.0001 vs. the control group.
Fig. 2. Seasonal changes in the birth to weaning period of the piglets. Data analysis was performed as described in Fig. 1. The error bars show mean ± SE. The piglet number can be referred from the data in Fig. 1a. Open circles indicate the data in the control group, while closed circles indicate the data in the compost extract-fed group. ⁄p < 0.05 and ⁄⁄⁄p < 0.005 (vs. control group).
preparation of the compost extract. In fact, Bacillus and Bacillus-related bacteria have been detected when the dehydrated compost was incubated on potato dextrose agar medium (Niisawa et al., 2008). Thus most microbes might survive as spore in the compost. We are now investigating whether these microbes in the compost reactivate in the gastrointestinal tract of mammals. The rate of stillbirth is higher in winter and lower in spring (Fig. 1c), probably due to season-associated climate stresses of
the sows (Tast et al., 2002; Renaudeau et al., 2003; Laws et al., 2009). Although similar seasonal changes in the rate of stillbirths were also observed in the compost extract-fed group, the rate was lower during all seasons. Therefore, administration of the compost extract may help to maintain health in the sows. In addition, the rate of stillbirth is significantly decreased in parities 7 and greater of the compost group in comparison with the control group (Table 3). A concomitant increase in pregnancy losses with aging of sows has been reported (Bouchard et al., 1995; Leenhouwers et al., 1999; Lucia Jr. et al., 2002). This may, at least in part, be due to greater obesity (Muirhead and Alexander, 1997), a prolonged farrowing due to weakened uterine muscle (Pejask, 1984), larger litters (Borges et al., 2005), and/or decline of maternal health with aging (Alonso-Spilsbury et al., 2005). It has been reported that pregnancy and lactation are physiological situations where major changes in energy homeostasis occur to meet the nutrient demands of foetal growth and milk production (Josephs et al., 2007). Maternal nutrition affects foetal growth and health of the offspring (Mahan and Vallet, 1997; Cerisuelo et al., 2009; Laws et al., 2009). Since the age-related abnormalities in sows should occur even in the compost extract-fed group, the prevention of stillbirths and the growth promotion of piglets observed in this study may be due to the improvement of the health of sows fed the compost extract. The health and milk quality of sows in the compost group were not investigated in this study. Finally it should be noted that the commercial feeds used in the farms contain probiotics, including Bacillus subtilis, Saccharomyces cerevisiae, and lactic acid bacteria. These probiotic mesophilic bacteria are involved in the prevention of gut disorders, and the bacteria in the compost extract may interact with these probiotic bacteria, resulting in an enhancement of the probiotic effects. Huang et al. (2008) reported that the spores of mesophilic Bacillus sp., such as B. subtilis, Bacillus licheniformis and Bacillus flexus, might activate innate immunity. This observation implicates the possible probiotic function of the spores of thermophilic Bacillus spp. Interestingly, B. subtilis has been isolated from the compost (Niisawa et al., 2008). Because the aqueous extract of the compost contains negligible nutrients, its thermophilic bacterial population (Niisawa et al., 2008) might be the cause of reduced stillbirths and growth promotion of piglets. The bacterial population formed by the
142
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
thermophilic composting process may be a useful source of probiotic bacteria for mammals. To our knowledge, these observations are the first showing that the oral administration of an aqueous extract of a thermophile-fermented compost affects physiological responses in a mammalian model. Thus the effects of the compost on the physiological conditions in the pigs and rodents are now under investigation. Acknowledgements This study was partly supported by a Grant-Aid for Research and Development Support Program for regional revitalization of the food industry sector of the Ministry of Agriculture, Forestry, and Fisheries of Japan. We thank Mr. Kazuo Ogawa (Keiyo Plant Engineering Co. Ltd.), Mr. Toshihito Shinmyou (Keiyo Plant Engineering Co. Ltd.) for sampling porcine fecal matter and checking data, Mr. Yoshifumi Kumagai (Keiyo Plant Engineering Co. Ltd.) for excellent technical assistance, and Makiko Hoshi-Miyamoto (Japan Eco-science Co. Ltd.) for excellent statistical analyses. References Alonso-Spilsbury, M., Monta-Rojas, D., Villanueva-Garcia, D., Martinez-Burnes, J., Orozco, H., Ramirez-Necoechea, R., Mayagoitia, A.L., Trujillo, M.E., 2005. Perinatal Asphyxia pathophysiology in pig and human: a review. Animal Reproduction Science 90, 1–30. Andersen, A.M., Vastrup, P., Wohlfahrt, J., Andersen, P.K., Olsen, J., Melbye, M., 2002. Fever in pregnancy and risk of stillbirth: a cohort study. Lancet 360, 1552–1556. Bäckhed, F., Ley, R.E., Sonnenburg, J.L., Gordon, J.I., 2005. Host-bacterial mutualism in the human intestine. Science 307, 1915–1920. Borges, V.F., Bernardir, M.L., Bortolozzo, F.P., Wentz, I., 2005. Risk factors for stillbirth and foetal mummification in four Brazilian swine herds. Preventive Veterinary Medicine 70, 165–176. Bouchard, G., McLaughlin, R.M., Ellersieck, M.R., Krause, G.F., Flanklin, C., Reddy, C.S., 1995. Retrospective evaluation of production characteristics in Sinclair miniature swine – 44 years later. Laboratory Animal Science 45, 408–414. Broom, L.J., Miller, H.M., Kerr, K.G., Knapp, J.S., 2006. Effects of zinc oxide and Enterococccus faecium SF68 dietary supplementation on the performance, intestinal microbiota and immune status of weaned piglets. Research in Veterinary Science 80, 45–54. Cerisuelo, A., Baucells, M.D., Gasa, J., Coma, J., Carrión, D., Chapinal, N., Sala, R., 2009. Increased sow nutrition during midgestation affects muscle fiber development and meat quality, with no consequences on growth performance. Journal of Animal Science 87, 729–739. Chen, K.L., Kho, W.L., You, S.H., Yeh, R.H., Tang, S.W., Hsieh, C.W., 2009. Effects of Bacillus subtilis var. natto and Saccharomyces cerevisiae mixed fermented feed on the enhanced growth performance of broilers. Poultry Science 88, 309–315. Davis, M.E., Parrott, T., Brown, D.C., de Rodas, B.Z., Johnson, Z.B., Maxwell, C.V., Rehberger, T., 2008. Effect of a Bacillus-based direct-fed microbial feed supplement on growth performance and pen cleaning characteristics of growing-finishing pigs. Journal of Animal Science 86, 1459–1467. Eckburg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E., Dethlefsen, L., Sargent, M., Gill, S.R., Nelson, K.E., Relman, D.A., 2005. Diversity of the human intestinal microbial flora. Science 308, 1635–1638. Foxcroft, G.R., 1997. Mechanisms mediating nutritional effects on embryonic survival in pigs. Journal of reproduction and fertility (Suppl. 52), 47–61. Fuller, R., 1991. Probiotics in human medicine. Gut 32, 439–442. Hong, H.A., Huang, J.M., Khaneja, R., Hiep, L.V., Urdaci, M.C., Cutting, S.M., 2008. The safety of Bacillus subtilis and Bacillus indicus as food probiotics. Journal of Applied Microbiology 105, 510–520.
Huang, J.M., La Ragione, R.M., Nunez, A., Cutting, S.M., 2008. Immunostimulatory activity of Bacillus spores. FEMS Immunology and Medical Microbiology 53, 195–203. Ishii, K., Takii, S., 2003. Comparison of microbial communities in four different composting processes as evaluated by denaturing gradient gel electrophoresis analysis. Journal of Applied Microbiology 95, 109–119. Josephs, T., Waugh, H., Kokay, I., Gratten, D., Thompson, M., 2007. Fasting-induced adipose factor identified as a key adipokine that is up-regulated in white adipose tissue during pregnancy and lactation in the rat. Journal of Endocrinology 194, 305–312. Kyriakis, S.C., Tsiloyiannis, V.K., Vlemmas, J., Sarris, K., Tsinas, A.C., Alexopoulos, C., Jansegers, L., 1999. The effect of probiotic LSP 122 on the control of postweaning diarrhoea syndrome of piglets. Research in Veterinary Science 67, 223–228. Laws, J., Litten, J.C., Laws, A., Lean, I.J., Dodds, P.F., Clarke, L., 2009. Effect of type and timing of oil supplements to sows during pregnancy on the growth performance and endocrine profile of low and normal birth weight offspring. British Journal of Nutrition 101, 240–249. Ley, R.E., Peterson, D.A., Gordon, J.I., 2006. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124, 837–848. Leenhouwers, J.I., Van der Lende, T., Knol, E.F., 1999. Analysis of stillbirth in different lines of pig. Livestock Production Science 57, 243–253. Leenhouwers, J.I., Wissink, T., van der Lende, T., Paidaans, H., Knol, E.F., 2003. Stillbirth in the pig in relation to genetic merit for farrowing survival. Journal of Animal Science 81, 2419–2424. Lucia Jr., T., Corrêa, M.N., Deschamps, J.C., Bianchi, I., Donn, M.A., Machado, A.C., Meincke, W., Matheus, J.E.M., 2002. Risk factors for stillbirths in two swine farms in the south of Brazil. Preventive Veterinary Medicine 53, 285–292. Mahan, D.C., Vallet, J.L., 1997. Vitamin and mineral transfer during foetal development and the early postnatal period in pigs. Journal of Animal Science 75, 2731–2738. Muirhead, M.R., Alexander, T.J.L., 1997. Managing pig health and the treatment of disease. A reference for the farm. 5M Enterprises, Sheffield, 133–226. Niisawa, C., Oka, S., Kodama, H., Hirai, M., Kumagai, Y., Mori, K., Matsumoto, J., Miyamoto, H., Miyamoto, H., 2008. Microbial analysis of composted product of marine animal resources and isolation of antagonistic bacteria to plant pathogen from the compost. The Journal of General and Applied Microbiology 54, 149–158. Pejask, Z., 1984. Some pharmacological methods to reduce intrapartum death of piglets. Pigs News Information 5, 35–37. Renaudeau, D., Anaïs, C., Noblet, J., 2003. Effects of dietary fiber on performance of multiparous lactating sows in a tropical climate. Journal of Animal Science 81, 717–725. Sakai, K., Narihara, M., Kasama, Y., Wakayama, M., Moriguchi, M., 1994. Purification and characterization of thermostable b-N-Acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost. Applied and Environmental Microbiology 60, 2911–2915. Sakai, K., Yokota, A., Kurokawa, H., Wakayama, M., Moriguchi, M., 1998. Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitin-containing compost. Applied and Environmental Microbiology 64, 3397–3402. Smith, G.C., Fretts, R.C., 2007. Stillbirth. Lancet 370, 1715–1725. Takaku, H., Kodaira, S., Kimoto, A., Nashimoto, M., Takagi, M., 2006. Microbial communities in the garbage composting with rice hull as an amendment revealed by culture-dependent and -independent approaches. Journal of Bioscience and Bioengineering 101, 42–50. Tanaka, R., Miyamoto, H., Kodama, H., Kawachi, N., Udagawa, M., Miyamoto, H., Matsushita, T., 2010. Feed additives with thermophile-fermented compost enhance concentrations of free amino acids in the muscle of the flatfish Paralichthys olivaceus. The Journal of General and Applied Microbiology 56, 61– 65. Tast, A., Peltoniemi, O.A., Virolainen, J.V., Lover, R.J., 2002. Early disruption of pregnancy as a manifestation of seasonal infertility in pigs. Animal Reproduction Science 15, 75–86. Zaleski, H.M., Hacker, R.R., 1993. Effect of oxygen and neostigmine on stillbirth and pig viability. Journal of Animal Science 71, 298–305.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
The oral administration of thermophile-fermented compost extract and its influence on stillbirths and growth rate of pre-weaning piglets Hirokuni Miyamoto a,b,⇑, Hiroaki Kodama b, Motoaki Udagawa c, Kenichi Mori a,b, Jiro Matsumoto d, Hatsumi Oosaki e, Tatsuo Oosaki e, Masayuki Ishizeki f, Daisuke Ishizeki f, Ryusuke Tanaka g, Teruo Matsushita g, Yuriko Kurihara h, Hisashi Miyamoto i a
Japan Eco-science Co. Ltd. (Nikkan Kagaku), 11-1-2F Shiomigaoka-chou, Chuou-ku, Chiba 260-0034, Japan Graduate School of Horticulture, Chiba University, 648 Matsudo, Chiba 271-8501, Japan Keiyo Plant Engineering Co. Ltd., 2-8-8 Ichikawaminami, Ichikawa-city, Chiba 272-0033, Japan d Keiyo Gas Co. Ltd., 2-8-8 Ichikawaminami, Ichikawa-city, Chiba 272-0033, Japan e Oosaki Swine Business Ltd., 1195-1 Takikubo-chou, Maebashi, Gunma 371-0235, Japan f Ishizeki Swine Farm Ltd., 1030 Komeno, Fujimimura, Seta, Gunma 371-0111, Japan g Department of Food Science and Technology, National Fisheries University, 2-7-1 Nagatahonchou, Shimonoseki, Yamaguchi 759-6595, Japan h Graduate School of Health Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyou-ku, Tokyo 113-8510, Japan i Miroku Co. Ltd., 706-27 Iwaya, Kitsuki, Oita 873-0021, Japan b c
a r t i c l e
i n f o
Article history: Received 16 October 2009 Accepted 20 June 2011
Keywords: Thermophile Compost Pig Foetal death Development
a b s t r a c t Food produced via fermentation with mesophilic bacteria has been used to confer health benefits. In contrast, mammalian physiological responses to the intake of thermophile-fermented products have not been thoroughly investigated. We examined the effects of administering a compost extract consisting of fermented marine animals with thermophiles, including Bacillaceae, to pregnant sows and piglets. Retrospective studies were performed on two different swine farms (n = 330–1050 sows). The rate of stillbirth was markedly lower in all parities of the compost extract-fed group compared to those of the control group (p 5 0.001). Additionally, the birth to weaning period of newborns was significantly shorter (p < 0.0001), while the ratio of weanlings per liveborn piglets was increased by more than 6.5% in the compost extract-fed group. Thus thermophiles and their products in the compost extract might promote growth and reduce stillbirths of piglets during the birth to weaning period. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The mammalian gastrointestinal tract is a complex ecological environment that is colonized with 100 trillion (1014) microorganisms (Ley et al., 2006), and dominated by members of the Bacteroidetes, Firmicutes, and Archaea (Bäckhed et al., 2005; Eckburg et al., 2005; Ley et al., 2006). Due to the diversity and complexity of these microorganisms, the host-microbe relationship has been poorly studied. Probiotics are live microorganisms, which, when ingested in sufficient amount, confer a health benefit to the host (Fuller, 1991; Kyriakis et al., 1999; Broom et al., 2006). Fermented foods and their included microbes have been commercialized as healthy food or feed and have multiple physiological effects on the health
Abbreviations: FAA, free amino acid; TDN, total digestible nutrients.
⇑ Corresponding author. Address: Japan Eco-science Co. Ltd., 11-1-2F Shiomigaoka-chou, Chuou-ku, Chiba-city, Chiba 260-0034, Japan. Tel.: +81 43 302 2322; fax: +81 43 302 2123. E-mail address: [email protected] (H. Miyamoto). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.06.018
of humans and livestock animals (Broom et al., 2006; Hong et al., 2008; Chen et al., 2009). Most probiotic microbes in fermented foods or feeds are mesophilic microbes. At present, it is not understood whether other microbes, e.g., extremophiles that contain Archaea and their metabolites, have probiotic effects on mammalian health. Recently, we reported the microbial diversity in a unique compost used as a fertilizer in Japan (Niisawa et al., 2008). This compost is a fermented marine animal resources by-product and includes small fish, crabs, and shrimps. These substrates are rapidly fermented at high temperature (approximately 75 °C) by fermentation-associated self-heating, and the final product contains several extremophiles, including thermophiles (Niisawa et al., 2008). The level of Firmicutes decreases during the maturation phase of fermentation in most other composts (Ishii and Takii, 2003; Takaku et al., 2006), but Firmicutes (mainly Bacillaceae) remain dominant in this compost. Strains with 16S rDNA sequences similar to those of Bacillus thermoamylovorans and Bacillus thermocloacae are the predominant bacteria in this compost (Niisawa et al., 2008). In addition, the compost contains a characteristic
138
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
bacterium that produces a cyclic lipopeptide with antifungal activity (Niisawa et al., 2008). Another research group has also reported the isolation of two or more bacteria that produce thermostable exo- and endo-chitinases in this compost (Sakai et al., 1994, 1998). Several Japanese farmers use this compost as an organic fertilizer. The compost is distributed over the soil, or its extract is sprayed onto the leaves of plants. Some of these plants have been used as a feed for chickens and pigs, and composting thermophilic bacteria on the plant surface are ingested by these animals. The thermophilic bacteria in the compost might affect the health of these animals in a direct and/or indirect manner. We initiated an investigation into the effects of the oral administration of an extract of this compost on several animal species. The addition of faeces from compost-fed pigs into the compost process of pig faeces increased the temperature of the compost by 10 °C over that achieved when faeces from noncompost-fed pigs was added. Fermentation at this higher temperature (60–90 °C) reduced the unpleasant odor and shortened the fermentation period. Thus, many Japanese stockbreeders for chickens or pigs now use the compost extract as a feed additive. In addition, this compost or its extract has been used as a feed additive for flatfish Paralichthys olivaceus in a farm and was reported to improve the health of the fish and raise the level of free amino acids in muscle tissue (Tanaka et al., 2010). In this paper, we identify additional effects of this compost extract based on observations of long-term administration to pigs, including prevention of stillbirth in sows and growth promotion in their piglets. To our knowledge, the study presented here is the first to investigate mammalian physiological responses to the oral administration of thermophile-fermented compost extract.
2. Materials and methods 2.1. Animals Sows (crossbred Landrace Large White) and their piglets (crossbred sows Duroc) were conventionally maintained at two Japanese farms (A and B). All pigs were fed a commercial feed (Table 1) and potable water ad libitum. Each sow and her piglets were housed in an individual pen (2.5 m 1.0 m) during the birth to weaning period. The housing area was maintained with 65–80% humidity and ventilation 0.6 m3/min. The temperature of the area was at 18–35 °C dependent on the seasonal changes. The individual piglets in each pen were selected at random and their daily body weights obtained. When the body weight of piglets reached 6.0–6.5 kg, the piglets were weaned and then housed together in another pen (2.5 m 1.8 m) (30 piglets per pen). All treatments of pigs on Farm A and Farm B (Gunma, Japan) were conducted in accordance with the institutional animal care guidelines for Oosaki Swine Business Ltd., Japan, and the Ishizeki Swine Farm Ltd., Japan, respectively. Sow management was according to individual farmspecific guidelines in order to most efficiently produce piglets (Leenhouwers et al., 2003). 2.2. Feeding conditions As seen in Table 1, sows were given two types (X and Y) of sowspecific commercial feed (Ryoumou Bussan Co. Ltd., Japan). The sows were usually given a restricted volume of feed Y (3–4 kg per day). From seven days before birth until all piglets were
Table 1 Composition of the diets in the farms. Sow feeda
Item
a
Piglet feedb
X
Y
I
II
III
IV
Component (%) Protein Lipid Fiber Ash Calcium Phosphate TDNc
17.00 4.00 4.00 6.50 0.85 0.65 77.00
12.00 2.50 4.50 6.00 0.80 0.65 74.00
18.50 5.00 3.00 8.50 0.65 0.55 83.50
18.50 4.00 3.00 8.50 0.65 0.55 80.00
20.50 4.50 3.00 6.50 0.70 0.06 79.00
13.50 4.00 4.00 5.00 0.55 0.40 78.00
Materials (%) Corn Soybeans Fish meal powder Rice bran Rice–lipid bran premixd Rice–wheat bran premixe Animal fat & feed additive premixf Non-animal fat & feed additive premixg Animal fat & lactose & feed additive premix Ah Animal fat & lactose & feed additive premix Bi Lactose & feed additive premixj Whey & fish meal premixk
63.27 27.00 2.00 0.00 0.00 2.03 5.70 0.00 0.00 0.00 0.00 0.00
76.32 15.50 0.00 0.00 0.00 0.93 0.00 8.15 8.15 0.00 0.00 0.00
44.00 21.00 0.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 5.71 18.00
54.00 24.00 0.00 1.00 0.00 0.00 0.00 0.00 11.00 0.00 0.00 10.00
59.77 32.50 2.00 0.00 0.02 0.00 0.00 0.00 0.00 5.71 0.00 0.00
74.38 21.00 0.00 0.00 0.07 0.00 4.55 0.00 0.00 0.00 0.00 0.00
Sows were given the sow feed X or sow feed Y (see Materials and methods). Piglets were given the piglet feeds, I, II, III, IV as described in Materials and methods. TDN shows total digestible nutrient. d Rice–lipid bran premix contains a mixture of rice bran and lipid bran. e Rice–wheat bran premix contains a mixture of rice bran and wheat bran. f Animal fat & feed additive premix contains a mixture of animal fat, calcium phosphate, calcium carbonate, salt, yucca foam extract, alfalfa meal, Bacillus subtilis, Saccharomyces cerevisiae, and lactic acid bacteria. g Non-animal fat & feed additive premix contains calcium phosphate, alfalfa meal, yucca foam extract, calcium carbonate, salt, B. subtilis, S. cerevisiae, and lactic acid bacteria. h The premix A contains a mixture of animal fat, lactose, calcium phosphate, calcium carbonate, yucca foam extract, citrate, lactate, salt, B. subtilis, S. cerevisiae, and lactic acid bacteria. i The premix B contains a mixture excluded S. cerevisiae from the premix A. j Lactose & feed additive premix contains a mixture of lactose, plant fat, calcium phosphate, calcium carbonate, yucca foam extract, citrate, lactate, salt, B. subtilis, S. cerevisiae, and lactic acid bacteria. k Whey & fish meal premix contains a mixture of whey and fish meal powder. b
c
139
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
weaned, sows were given 5–6 kg per day of feed X instead of feed Y. Piglets were given four types of commercial feed (I–IV, see Table 1). From three to 28 days after birth (during the birth to weaning period and about 5–10 days after weaning), piglets were provided feed I (Nisshin-Marubeni Co. Ltd., Japan). After weaning, the piglets (from 28 to 34 days after birth) were fed with feed II (Nisshin-Marubeni Co. Ltd., Japan). From 35 days after birth until the body weight of the piglets exceeded 30 kg, they were fed feed III (Ryoumou Bussan Co. Ltd., Japan). Thereafter, piglets (body weight between 30 kg and 70 kg) were provided feed IV (Ryoumou Bussan Co. Ltd., Japan). 2.3. Treatment of compost The compost used in this study was made from marine animals and coffee residues by a repeated fed-batch fermentation system with three bioreactors, as described in our recent paper (Niisawa et al., 2008). Bacteria in this compost were mainly Bacillaceae, and the 16S rDNA sequences of major bacterial species showed high similarity to those of B. thermoamylovorans and B. thermocloacae. This compost has been marketed as an organic fertilizer or a fermentation feed for pigs and chickens (Miroku Co. Ltd., and Keiyo Plant Engineering Co. Ltd., Japan). The compost was diluted to 1/ 100 with potable water (as v/v), and the resulting suspension incubated aerobically at 60 °C for 10–24 h. The compost suspension was then filtered through a nylon mesh (pore size 100 lm). This compost extract (pH 7.5–8.5) contained <99.9% H2O, <0.1% protein, <0.1% lipid, <0.1% fiber, <0.1% ash, <50 ppm lactate, and <50 ppm butyrate, indicating that the compost extract itself included negligible nutrients. The number of bacteria in the compost extract was assessed as <5.0 106 CFU/ml thermophiles, after culture on nutrient agar (1% beef extract, 1% peptone, and 0.5% NaCl) at 55 °C. The administered aqueous solution of 0.4% (v/v) compost extract was supplied as drinking water to the pigs. After dilution, the water contained <0.2 ppm butyrate and lactate. On Farm A, all pigs housed from February 2004 to the end of 2007 were fed the extract. When administration of the compost extract began in February 2004, the concentration was gradually increased from 0.2% to 0.4% (0.1% increment per week). During the three-year period, the supply of the compost extract was interrupted for one month in October 2004, due to maintenance of the water supply system. All pigs on Farm A were housed from January 2001 to January 2004 and supplied compost extract-free potable water. On Farm B, all pigs housed from July 2006 to June 2008 were provided an aqueous water solution containing 0.4% (v/v) compost extract. Group B pigs, housed from January 2004 to June 2006 were provided compost extract-free potable water. Farms A and B are in the same area of Gunma Prefecture (10 km distance between these two Farms). While the pigs in Farm A were supplied with the compost extracts (2004–2007), the number of stillbirths under the compost extract-free condition was investigated at Farm B (2004–2006). Thus, the effects of climatic conditions between 2004–2006 on stillbirths would be insignificant. 2.4. Data analyses In order to determine which non-parametric or parametric statistical analyses are suitable to all subjects, P values were calculated by using the Kolmogorov–Smirnov test. When the P values were less than 0.05, a non-parametric statistic analysis was suitable for comparisons between two subjects. Here all the data were statistically analyzed using the Mann–Whitney U test (Wilcoxon signed-rank test) (one category in the control group vs. the same category in the compost extract group housed on the same farm). All data were hypothesized as individual subjects, since the body condition of sows varied. Statistical analyses were carried out with
StatView-J 4.02 software for Macintosh. All data are expressed as mean ± SE. 3. Results 3.1. Effects of the thermophile compost on foetal death of piglets The effects of thermophile compost as a feed additive for pigs on foetal deaths were investigated (Table 2). On Farm A, the number of newborn piglets per sow did not differ between the sows administered potable water (control group) and those administered compost solution (compost group). The number of liveborn piglets per sow tended to be higher in the compost-group than the control group (p = 0.082; vs. control-group). A significant difference was obtained for the number of stillbirths (p < 0.0001; vs. control group) and for the rate of stillbirths (p < 0.0001). The number of stillbirths per delivery on Farm B was also significantly reduced (p = 0.0006; vs. control group) (Table 2), showing a significant reduction in the rate of stillbirths in the compost group (p = 0.001; vs. control group). In Farm B, the total number of newborn piglets per sow slightly decreased (p = 0.0039; vs. control group), although the number of liveborn piglets in the compost group was mostly unchanged from that of the control group. Thus, these data suggest that the oral administration of the compost solution to sows might reduce stillbirths. 3.2. Seasonal changes in the rate of stillbirth We detected whether seasonal changes in the stillbirth rate were affected by administration of the compost solution. As seen in Fig. 1a and b, 12–15 sows farrowed, and 150–170 piglets were born every month on Farm A. The stillbirth rate was high in the winter and gradually decreased in the spring in the control group. These seasonal changes in the rate of stillbirths were also evident in the compost group. The lowest rate of stillbirths was seen in early spring (March–April). Each monthly rate of stillbirths between the control and compost groups tended to be different (p < 0.1), except for the period from November to December. Similar results were observed on Farm B (data not shown). These
Table 2 Stillbirth rates for two different farms. Category
Control group
Compost group
Farm A Total number of sows Total number of newborns Total number of stillbirths Newborn piglets per sow Liveborn piglets per sow Stillbirth per sow Rate of stillbirth (%)
986 11,568 1386 11.74 ± 0.10 10.34 ± 0.09 1.41 ± 0.06 11.74 ± 0.10
1050 12,171 1055 11.59 ± 0.11 10.58 ± 0.11a 1.01 ± 0.05b 8.60 ± 0.40b
Farm B Total number of sows Total number of newborns Total number of stillbirths Newborn piglets per sow Liveborn piglets per sow Stillbirth per sow Rate of stillbirth (%)
330 3906 596 11.84 ± 0.18 10.34 ± 0.09 1.81 ± 0.11 15.41 ± 1.05
421 4707 565 11.18 ± 0.15c 9.84 ± 0.14 1.34 ± 0.08d 11.42 ± 0.69e
In Farm A, births were recorded from January 2001 until January 2004, (control group) and from February 2004 until February 2007 (compost group). In Farm B, births were recorded from July 2004 until June 2006 (control group) and from July 2006 until June 2008 (compost group). All data are shown as the mean ± SE. a p < 0.1. b p < 0.0001. c p < 0.005. d p < 0.001. e p < 0.01 (vs. control group).
140
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
iod (three years). The control group showed a lower value for the average of parity than the compost group (3.55 vs. 4.15 in Farm A; 3.39 vs. 3.98 in Farm B). Although the sows of the control group have a potential for showing a lower stillbirth rate than the sows of the compost group with respect to the number of pregnancies, the results showed a marked reduction in the stillbirth rate of the compost group. 3.4. Effects of the thermophile compost on the growth of piglets during birth to weaning The effects of administration of the compost extract to sows on the growth of their offspring during the lactation period were investigated on Farm A (n = 9290–10,403) (Table 4). The average number of weanlings per sow was increased by about 4.7% in the compost group compared to that of the control group (p < 0.0001). In addition, the ratio of weanlings to liveborn piglets also increased by about 6.5% in the compost group (p < 0.0001). Therefore, the oral administration of the compost solution to the lactating sow apparently increased the health of the piglets during birth to weaning. The body weights of the piglets just after weaning were similar in the two groups. It is worthwhile to note that the lactation period was shortened by about three days in the compost group in comparison to the control group (p < 0.0001). The seasonal trend in weaning duration is shown in Fig. 2. The lactation period was shorter in the summer than in the winter, as seen in the control group. The lactation period of the compost group in the winter was nearly the same as that of the control group in the summer, indicating that the administration of the compost extract to the sow enhanced the growth of their piglets during the lactation period. This duration was significantly shortened (p < 0.0001), especially in January and February (p < 0.05), May and June (p < 0.05), July and August (p < 0.05), and November and December (p < 0.0001). Thus, administration of the compost extract to sows enhanced the growth of their piglets until weaning. 4. Discussion and conclusions
Fig. 1. Seasonal changes in number of sows (a), number of newborns (b), and rate of stillbirth (c) in Farm A. The year was divided into six periods of two months each. The data were obtained by counting pig numbers for three years. Since the counting of newborns, their sows, and stillbirth rate was carried out every month, averages were calculated for a two-month unit (n = 6 per period, except January–February (n = 7)). The error bars show mean ± SE. Open circles indicate the data in the control group, while closed circles indicate the data in the compost extract-fed group. ⁄ p < 0.05, and ⁄⁄p < 0.01 (vs. control group).
observations indicate that the rate of stillbirths of the sows of the compost group was stable at a low level during all seasons. 3.3. Effects of sow pregnancy number on stillbirth rates of their piglets We investigated the relationship between parity and stillbirth rate on Farms A and B. The stillbirth rate increased according to the increase in parities in both groups. However, the stillbirth rate in the compost group was markedly lower for all parities when compared with that of the control group (Table 3). The stillbirth rate significantly increased in the control sows of parities 7 and greater, but the increase in stillbirth rate was significantly lower in the compost group. These observations suggest that the increase in stillbirth rate according to the increase in parities tended to be alleviated by administration of the compost extract to sows. This result indicates the importance of parity. We determined the average of parity in both groups during the experimental per-
In this paper, the physiological effects of the oral administration of the extract of thermophile-fermented compost to pigs are addressed in regard to the stillbirth rate of newborns and growth of the piglet. The retrospective studies indicated reduction in the stillbirth rate at two different swine farms where the compost solution was administered. The stillbirth rate in the compost group was markedly lower in all parities of sows compared to the control group. The shorter lactation period in the compost group indicates growth promotion of piglets in addition to the increased number of weaned piglets. Taken together, the preservation and/or improvement of the healthy condition of the sows are important for normal growth of fetuses and newborns (Foxcroft, 1997), and the intake of thermophile-fermented compost extract is apparently beneficial for sustainment of the healthy condition of the host. Possible risk factors for stillbirth have been surveyed (Zaleski and Hacker, 1993; Andersen et al., 2002; Leenhouwers et al., 2003; Smith and Fretts, 2007), but factors that reduce the stillbirth rate have rarely been reported. Recently it has been reported that a microbial feed, which contain mesophilic Bacillus species, reduced mortality of pigs during the growing-finishing period. (Davis et al., 2008). The approximately 570-bp-long 16S rDNA sequences that had been amplified from total DNA of the bacteria in the compost were determined, and the results indicated that the spore-forming bacteria such Bacillus or Bacillus-related genera were predominant in the compost (Niisawa et al., 2008). Most of these bacteria form endospores, and thus pigs might ingest spores in addition to vegetative forms. Compost is preserved in a desiccated condition until
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
141
Table 3 Relationship between parities and stillbirth rates for Farm B. Parities
Control group
Compost group
Number of sows
Stillbirth rate
Number of sows
Stillbirth rate
Farm A 1–3 4–6 7 and greater
416 248 101
12.00 ± 0.51 13.61 ± 0.62 16.57 ± 1.12a,b
269 203 134
8.52 ± 0.59c 10.14 ± 0.69d,e 12.33 ± 0.88f,g
Farm B 1–3 4–6 7 and greater
171 172 67
11.74 ± 1.02 15.13 ± 1.19 27.85 ± 3.29b,h
143 149 112
9.01 ± 1.24i 11.35 ± 1.03j 14.92 ± 1.43k,l
The parities express the number of times the sow had been pregnant. All data are shown as the mean ± SE. a p < 0.001 vs. parities 1–3 for the control group. b p < 0.01 vs. parities 4–6 within the same group. c p < 0.0001 vs. parities 1–3 in the control group. d p < 0.0001 vs. parities 4–6 within the compost group. e p < 0.001 vs. parities 1–3 in the same group. f p < 0.001 vs. parities 7 and greater in the control group. g p < 0.0001 vs. parities 1–3 in the same group. h p < 0.0001 vs. parities 1–3 in the control group. i p < 0.1 vs. parities 1–3 in the control group. j p < 0.05 vs. parities 4–6 in the control group. k p < 0.01 vs. parities 1–3 within the compost group. l p < 0.001 vs. parities 7 and greater in the control group.
Table 4 Ratio of weanlings and the lactation period. Category
Control group
Compost group
Total number of weanlings Number of weanlings per sow Weanlings per liveborn piglets (%) Duration of the lactation period (days)
9290 9.44 ± 0.08 87.02 ± 1.52 24.63 ± 0.35
10,403 9.88 ± 0.08a 93.56 ± 0.51a 21.84 ± 0.27a
These data were obtained for Farm A (2001–2007). The numbers corresponding to total newborns (including living and dead) and liveborn piglets are shown in Table 1. All data are shown as the mean ± SE. a p < 0.0001 vs. the control group.
Fig. 2. Seasonal changes in the birth to weaning period of the piglets. Data analysis was performed as described in Fig. 1. The error bars show mean ± SE. The piglet number can be referred from the data in Fig. 1a. Open circles indicate the data in the control group, while closed circles indicate the data in the compost extract-fed group. ⁄p < 0.05 and ⁄⁄⁄p < 0.005 (vs. control group).
preparation of the compost extract. In fact, Bacillus and Bacillus-related bacteria have been detected when the dehydrated compost was incubated on potato dextrose agar medium (Niisawa et al., 2008). Thus most microbes might survive as spore in the compost. We are now investigating whether these microbes in the compost reactivate in the gastrointestinal tract of mammals. The rate of stillbirth is higher in winter and lower in spring (Fig. 1c), probably due to season-associated climate stresses of
the sows (Tast et al., 2002; Renaudeau et al., 2003; Laws et al., 2009). Although similar seasonal changes in the rate of stillbirths were also observed in the compost extract-fed group, the rate was lower during all seasons. Therefore, administration of the compost extract may help to maintain health in the sows. In addition, the rate of stillbirth is significantly decreased in parities 7 and greater of the compost group in comparison with the control group (Table 3). A concomitant increase in pregnancy losses with aging of sows has been reported (Bouchard et al., 1995; Leenhouwers et al., 1999; Lucia Jr. et al., 2002). This may, at least in part, be due to greater obesity (Muirhead and Alexander, 1997), a prolonged farrowing due to weakened uterine muscle (Pejask, 1984), larger litters (Borges et al., 2005), and/or decline of maternal health with aging (Alonso-Spilsbury et al., 2005). It has been reported that pregnancy and lactation are physiological situations where major changes in energy homeostasis occur to meet the nutrient demands of foetal growth and milk production (Josephs et al., 2007). Maternal nutrition affects foetal growth and health of the offspring (Mahan and Vallet, 1997; Cerisuelo et al., 2009; Laws et al., 2009). Since the age-related abnormalities in sows should occur even in the compost extract-fed group, the prevention of stillbirths and the growth promotion of piglets observed in this study may be due to the improvement of the health of sows fed the compost extract. The health and milk quality of sows in the compost group were not investigated in this study. Finally it should be noted that the commercial feeds used in the farms contain probiotics, including Bacillus subtilis, Saccharomyces cerevisiae, and lactic acid bacteria. These probiotic mesophilic bacteria are involved in the prevention of gut disorders, and the bacteria in the compost extract may interact with these probiotic bacteria, resulting in an enhancement of the probiotic effects. Huang et al. (2008) reported that the spores of mesophilic Bacillus sp., such as B. subtilis, Bacillus licheniformis and Bacillus flexus, might activate innate immunity. This observation implicates the possible probiotic function of the spores of thermophilic Bacillus spp. Interestingly, B. subtilis has been isolated from the compost (Niisawa et al., 2008). Because the aqueous extract of the compost contains negligible nutrients, its thermophilic bacterial population (Niisawa et al., 2008) might be the cause of reduced stillbirths and growth promotion of piglets. The bacterial population formed by the
142
H. Miyamoto et al. / Research in Veterinary Science 93 (2012) 137–142
thermophilic composting process may be a useful source of probiotic bacteria for mammals. To our knowledge, these observations are the first showing that the oral administration of an aqueous extract of a thermophile-fermented compost affects physiological responses in a mammalian model. Thus the effects of the compost on the physiological conditions in the pigs and rodents are now under investigation. Acknowledgements This study was partly supported by a Grant-Aid for Research and Development Support Program for regional revitalization of the food industry sector of the Ministry of Agriculture, Forestry, and Fisheries of Japan. We thank Mr. Kazuo Ogawa (Keiyo Plant Engineering Co. Ltd.), Mr. Toshihito Shinmyou (Keiyo Plant Engineering Co. Ltd.) for sampling porcine fecal matter and checking data, Mr. Yoshifumi Kumagai (Keiyo Plant Engineering Co. Ltd.) for excellent technical assistance, and Makiko Hoshi-Miyamoto (Japan Eco-science Co. Ltd.) for excellent statistical analyses. References Alonso-Spilsbury, M., Monta-Rojas, D., Villanueva-Garcia, D., Martinez-Burnes, J., Orozco, H., Ramirez-Necoechea, R., Mayagoitia, A.L., Trujillo, M.E., 2005. Perinatal Asphyxia pathophysiology in pig and human: a review. Animal Reproduction Science 90, 1–30. Andersen, A.M., Vastrup, P., Wohlfahrt, J., Andersen, P.K., Olsen, J., Melbye, M., 2002. Fever in pregnancy and risk of stillbirth: a cohort study. Lancet 360, 1552–1556. Bäckhed, F., Ley, R.E., Sonnenburg, J.L., Gordon, J.I., 2005. Host-bacterial mutualism in the human intestine. Science 307, 1915–1920. Borges, V.F., Bernardir, M.L., Bortolozzo, F.P., Wentz, I., 2005. Risk factors for stillbirth and foetal mummification in four Brazilian swine herds. Preventive Veterinary Medicine 70, 165–176. Bouchard, G., McLaughlin, R.M., Ellersieck, M.R., Krause, G.F., Flanklin, C., Reddy, C.S., 1995. Retrospective evaluation of production characteristics in Sinclair miniature swine – 44 years later. Laboratory Animal Science 45, 408–414. Broom, L.J., Miller, H.M., Kerr, K.G., Knapp, J.S., 2006. Effects of zinc oxide and Enterococccus faecium SF68 dietary supplementation on the performance, intestinal microbiota and immune status of weaned piglets. Research in Veterinary Science 80, 45–54. Cerisuelo, A., Baucells, M.D., Gasa, J., Coma, J., Carrión, D., Chapinal, N., Sala, R., 2009. Increased sow nutrition during midgestation affects muscle fiber development and meat quality, with no consequences on growth performance. Journal of Animal Science 87, 729–739. Chen, K.L., Kho, W.L., You, S.H., Yeh, R.H., Tang, S.W., Hsieh, C.W., 2009. Effects of Bacillus subtilis var. natto and Saccharomyces cerevisiae mixed fermented feed on the enhanced growth performance of broilers. Poultry Science 88, 309–315. Davis, M.E., Parrott, T., Brown, D.C., de Rodas, B.Z., Johnson, Z.B., Maxwell, C.V., Rehberger, T., 2008. Effect of a Bacillus-based direct-fed microbial feed supplement on growth performance and pen cleaning characteristics of growing-finishing pigs. Journal of Animal Science 86, 1459–1467. Eckburg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E., Dethlefsen, L., Sargent, M., Gill, S.R., Nelson, K.E., Relman, D.A., 2005. Diversity of the human intestinal microbial flora. Science 308, 1635–1638. Foxcroft, G.R., 1997. Mechanisms mediating nutritional effects on embryonic survival in pigs. Journal of reproduction and fertility (Suppl. 52), 47–61. Fuller, R., 1991. Probiotics in human medicine. Gut 32, 439–442. Hong, H.A., Huang, J.M., Khaneja, R., Hiep, L.V., Urdaci, M.C., Cutting, S.M., 2008. The safety of Bacillus subtilis and Bacillus indicus as food probiotics. Journal of Applied Microbiology 105, 510–520.
Huang, J.M., La Ragione, R.M., Nunez, A., Cutting, S.M., 2008. Immunostimulatory activity of Bacillus spores. FEMS Immunology and Medical Microbiology 53, 195–203. Ishii, K., Takii, S., 2003. Comparison of microbial communities in four different composting processes as evaluated by denaturing gradient gel electrophoresis analysis. Journal of Applied Microbiology 95, 109–119. Josephs, T., Waugh, H., Kokay, I., Gratten, D., Thompson, M., 2007. Fasting-induced adipose factor identified as a key adipokine that is up-regulated in white adipose tissue during pregnancy and lactation in the rat. Journal of Endocrinology 194, 305–312. Kyriakis, S.C., Tsiloyiannis, V.K., Vlemmas, J., Sarris, K., Tsinas, A.C., Alexopoulos, C., Jansegers, L., 1999. The effect of probiotic LSP 122 on the control of postweaning diarrhoea syndrome of piglets. Research in Veterinary Science 67, 223–228. Laws, J., Litten, J.C., Laws, A., Lean, I.J., Dodds, P.F., Clarke, L., 2009. Effect of type and timing of oil supplements to sows during pregnancy on the growth performance and endocrine profile of low and normal birth weight offspring. British Journal of Nutrition 101, 240–249. Ley, R.E., Peterson, D.A., Gordon, J.I., 2006. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124, 837–848. Leenhouwers, J.I., Van der Lende, T., Knol, E.F., 1999. Analysis of stillbirth in different lines of pig. Livestock Production Science 57, 243–253. Leenhouwers, J.I., Wissink, T., van der Lende, T., Paidaans, H., Knol, E.F., 2003. Stillbirth in the pig in relation to genetic merit for farrowing survival. Journal of Animal Science 81, 2419–2424. Lucia Jr., T., Corrêa, M.N., Deschamps, J.C., Bianchi, I., Donn, M.A., Machado, A.C., Meincke, W., Matheus, J.E.M., 2002. Risk factors for stillbirths in two swine farms in the south of Brazil. Preventive Veterinary Medicine 53, 285–292. Mahan, D.C., Vallet, J.L., 1997. Vitamin and mineral transfer during foetal development and the early postnatal period in pigs. Journal of Animal Science 75, 2731–2738. Muirhead, M.R., Alexander, T.J.L., 1997. Managing pig health and the treatment of disease. A reference for the farm. 5M Enterprises, Sheffield, 133–226. Niisawa, C., Oka, S., Kodama, H., Hirai, M., Kumagai, Y., Mori, K., Matsumoto, J., Miyamoto, H., Miyamoto, H., 2008. Microbial analysis of composted product of marine animal resources and isolation of antagonistic bacteria to plant pathogen from the compost. The Journal of General and Applied Microbiology 54, 149–158. Pejask, Z., 1984. Some pharmacological methods to reduce intrapartum death of piglets. Pigs News Information 5, 35–37. Renaudeau, D., Anaïs, C., Noblet, J., 2003. Effects of dietary fiber on performance of multiparous lactating sows in a tropical climate. Journal of Animal Science 81, 717–725. Sakai, K., Narihara, M., Kasama, Y., Wakayama, M., Moriguchi, M., 1994. Purification and characterization of thermostable b-N-Acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost. Applied and Environmental Microbiology 60, 2911–2915. Sakai, K., Yokota, A., Kurokawa, H., Wakayama, M., Moriguchi, M., 1998. Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitin-containing compost. Applied and Environmental Microbiology 64, 3397–3402. Smith, G.C., Fretts, R.C., 2007. Stillbirth. Lancet 370, 1715–1725. Takaku, H., Kodaira, S., Kimoto, A., Nashimoto, M., Takagi, M., 2006. Microbial communities in the garbage composting with rice hull as an amendment revealed by culture-dependent and -independent approaches. Journal of Bioscience and Bioengineering 101, 42–50. Tanaka, R., Miyamoto, H., Kodama, H., Kawachi, N., Udagawa, M., Miyamoto, H., Matsushita, T., 2010. Feed additives with thermophile-fermented compost enhance concentrations of free amino acids in the muscle of the flatfish Paralichthys olivaceus. The Journal of General and Applied Microbiology 56, 61– 65. Tast, A., Peltoniemi, O.A., Virolainen, J.V., Lover, R.J., 2002. Early disruption of pregnancy as a manifestation of seasonal infertility in pigs. Animal Reproduction Science 15, 75–86. Zaleski, H.M., Hacker, R.R., 1993. Effect of oxygen and neostigmine on stillbirth and pig viability. Journal of Animal Science 71, 298–305.