Soy Phytoestrogens: Effects on litter size in swine

Todd A. Winters, William J. Banz, Elaine M. Carnevale, and Robert D. Arthur

Department of Animal Science, Food and Nutrition

Southern Illinois University

Carbondale, IL 62901

Technical Summary

Soybean meal contain estrogenic compounds called phytoestrogens that have been shown to effect reproductive function in animals. Soybean meal makes up a significant percentage (20%) of most swine diets. This research is concentrating on the effects of these compounds on swine reproduction. In vitro studies with swine ovarian granulosa cells indicate that phytoestrogens have the ability to decrease follicular cell atresia potentially leading to more follicle recruitment and ovulations, thus potentially increasing litter size. A swine feeding study is underway. Prepubertal gilts were assigned to two diet treatment groups: 1) standard soybean meal; and 2) low phytoestrogen soy protein concentrate. Parameters under examination are body weight, feed consumption, backfat, serum reproductive hormones, vulva diameters, first estrus, estrous cycle length, success of breeding, and litter size. Body weight, crown-rump length, and anogenital distance will be measured on offspring within 24 h of farrowing. Breeding success and litter size will then be examined for two subsequent gestations to determine if the possible effects of phytoestrogens are permanent or only occur when the animals are fed these soy compounds. Reproductive performance of offspring will also be followed over the following year. Since swine also have a great potential for being a model for human cardiovascular health studies, plasma HDL, LDL, total cholesterol, triglycerides, glucose, and insulin are also being measured. First trial gilts are just farrowing and two other trials are underway. Prepubertal, preliminary results show increases in frame (length, p<0.05; girth p<0.10) as well as an increase (p<0.05) in blood glucose for the soybean meal fed animals suggesting a phytoestrogen effect on growth and metabolism. Preliminary results on reproductive parameters suggest that the phytoestrogens had no effect on age at puberty, and suggest that estrous cycle length may be slightly increased in the phytoestrogen diet. Breeding success is mixed, and littersize is under examination. This project is ongoing, and since the initial IPPA support has received additional support from the Illinois Council on Food and Agricultural Research (C-FAR), Archer Daniels Midland Co., and the Illinois Soybean Checkoff Board.

Final Progress Report

Introduction

Soy products contain estrogenic compounds called phytoestrogens that have been shown to effect reproductive function in animals^(1,2,3). The isoflavones, Genistein and Daidzein, are the most common phytoestrogen in soybeans^(1,2) and remains in the meal after processing. Soybean meal makes up a significant percentage (20%) of most swine diets. Since soybean and pork production are two of the major agricultural industries in Illinois, the proposed research examining the effects of a soybean product on swine reproduction directly affects agriculture in the state. If soy products are to attain their full potential, a greater understanding of specific attributes of individual soy phytoestrogens must be developed. This project has concentrated on the effects of these compounds on swine reproduction.

Estrogens have been shown to affect ovarian function and early embryonic development. In swine, estradiol has been found to increase steroidogenesis in ovarian granulosa cells⁽⁴⁾, and in rats, decrease apoptosis (programmed cell death) of ovarian cells⁽⁵⁾. A decrease in ovarian follicle apoptosis could increase ovulation rate. Increased ovulation rate in swine (if coupled with increased rates of embryo survival) makes possible an increase in average litter size. Estrogens also play an important role in early embryonic development in swine. Specific examples of estrogen-regulated events occurring early in porcine pregnancy include intrauterine migration and spacing, morula-blastocyst transition, and maternal recognition of pregnancy⁽⁶⁾. Studies from our laboratory have shown that rats fed a diet high in genistein exhibited antagonistic effects on the uterus ⁽⁷⁾. This observation may have implications in inhibiting spontaneous abortions. Conversely, our research group has also found that a high-isoflavone clover diet in anestrous mares is agonistic to the uterus⁽⁸⁾. Such an effect would be detrimental to a pregnant animal, but may be species specific. The positive influence of phytoestrogens on these events during pregnancy would increase embryonic survival and thus litter size. An average increase of one pig per litter nationwide would allow us to produce 17 million more market pigs to meet expanding world needs (i.e., export market). In addition, a positive influence of soy phytoestrogens on reproduction will increase demand to isolate these compounds.

Evidence suggests that phytoestrogens may affect the female reproductive system, a primary target of estrogens^(1,2,3). Only one study has reported possible effects of soy phytoestrogens on reproduction in swine⁽³⁾. This study examined the effects of soybean meal on the reproductive development of prepubertal gilts. Although, estrogenic effects were observed (increased vulva diameters), results were inconclusive. Therefore a more comprehensive study of the effects of soy phytoestrogens on swine reproduction is warranted. Initial in vitro studies from our laboratory suggest that the soy phytoestrogens, genistein and daidzein, decrease ovarian follicle cell apoptosis in a dose responsive manner ^(9,10).

Estrogen has been implicated in the proper development of the reproductive tract because of known permanent reproductive alterations, including cancer, caused by fetal exposure to the synthetic estrogen, diethylstilbestrol (DES)⁽¹¹⁾. These defects suggest that tissues derived from the reproductive system progenitors were affected. Phytoestrogens may have a protective effect against estrogenic compounds like DES by binding to estrogen receptors, thus blocking the proliferative effects of stronger estrogens that may have deleterious effect on proper differentiation. This estrogen binding hypothesis has been proposed as the protective effect that soy-related phytoestrogens have on endocrine-related cancers (e.g. breast & prostate cancer)⁽¹⁾. Initial in vitro studies in our laboratory indicate that soy phytoestrogens affect cell division in porcine reproductive progenitor cells (M. Williams & T. Winters, unpublished).

The purpose of this research has been to determine what effects soy phytoestrogens have on the reproductive life cycle of the gilt and sow. It is possible that at certain times these soy compounds may have beneficial effects (i.e. ovulation rate), where at others times these compounds may be detrimental to reproduction and/or lactation. This information could lead to a better knowledge of possible feeding strategies in growing, breeding, gestating, and lactating gilts and sows.

Objective

Our overall objective was to determine the effects of soy-related phytoestrogens on swine reproduction. Specific aims are:

1. Effects of Ingested Soy Phytoestrogens on Fertility and Litter Size

To determine if soy phytoestrogens fed to gilts prepuberty through gestation positively affect breeding and/or increase litter sizes.

2. Long Term Effects of Soy Phytoestrogens on Swine Reproduction

To determine if soy phytoestrogens affect subsequent reproduction in sows or in their offspring that were exposed to these soy phytoestrogens in utero.

Achieving these objectives will be economically beneficial to swine and soybean producers by making pork production more efficient and by increasing the utilization and/or demand of soy products in the swine industry.

Methods

All animals have been produced and housed at the Southern Illinois University - Carbondale Farms. At 12 weeks of age, gilts (11-12 per treatment group) have been blocked by litter and randomly assigned to one of two diets: (1) Ethanol-washed soybean protein concentrate diet low in isoflavones (ADM, Decataur, IL) and (2) a standard soybean meal diet high in isoflavones. Experimental diets contain 15% crude protein by weight. Essential amino acids were balanced and adjusted on an energy basis equivalent according to the National Research Council. Animals have been allowed free access to their assigned diet and water over the experimental period.

Biweekly body weights have been recorded for the duration of the study. Backfat, length and girth measurements have been taken monthly. Blood samples have been taken monthly via jugular puncture for baseline measurement of serum reproductive steroids (progesterone, estrogen, testosterone), gonadotropins (LH, FSH), and phytoestrogens. The horizontal and vertical diameter of each vulva have been measured each week using calipers as a measurement of estrogenic stimulation⁽³⁾. Signs of behavioral estrus have been observed twice daily and noted as the gilts approach puberty. The gilts have continued on the diets through breeding and pregnancy. Success of breeding (services/conception) and litter size have been noted. Body weight, crown-rump measurements, and anogenital distance measurements⁽¹²⁾ have been taken on offspring within 24 h of farrowing. At castration, the testes of males are weighed and examined for gross morphological differences. The testes are then processed for later histological and chemical analyses in a collaborative project with Dr. Rex Hess of the University of Illinois who is interested in the effects of estrogenic compounds on fetal and neonatal testicular development. The sows on the diets are returned to a standard soybean meal diet at weaning. Breeding success and litter size will then be examined and analyzed in two subsequent gestations to determine if the possible effects of phytoestrogens are permanent or only occur when the animals are fed these soy compounds. Reproductive performance of offspring will also be followed over the following year.

Swine also have a great potential for being a model for human cardiovascular health studies. Soy phytoestrogens have been suggested to have a protective effect against cardiovascular disease⁽¹⁾. Therefore as a secondary objective, we have been examining certain parameters related to cardiovascular health. These include plasma cholesterol, triglycerides, glucose, and insulin.

Results and Discussion

First trial gilts are just farrowing and two other trials are underway. Preliminary results in prepubertal animals from the first trial show increases in frame (length, p<0.05; girth p<0.10) as well as an increase (p<0.05) in blood glucose for the soybean meal fed animals. Although trends were not significant, backfat appeared to be lower in soybean meal fed animals than in the low isoflavone diet. These initial results suggest a phytoestrogen effect on lean growth and metabolism. Genistein is known to inhibit glucose transport into the body's cells which may explain the increases in blood glucose⁽¹³⁾. Insulin measurements will determine the efficiency of insulin-stimulated glucose transport under the influence of soy isoflavones. Rat studies, from our research group, have found that a high isoflavone diet lowers the insulin/glucose ratio which leads to a leaner animal⁽¹⁴⁾. Such an effect would also be cardioprotective.

Preliminary results on reproductive parameters suggest that the phytoestrogens had no effect on age at puberty, and suggest that estrous cycle length may be slightly increased in the phytoestrogen diet. Menstrual cycles in women have been reported to be increased in women on high isoflavone diets⁽¹⁾. Breeding success is mixed, and littersize is under examination. Hormone assays have not been performed, and other reproductive parameters will be followed over the next two years in trial one, as well as trials two and three.

Leveraged Funding

Since the initial IPPA support, the team of investigators has received additional support from the Illinois Council on Food and Agricultural Research (C-FAR), Archer Daniels Midland Co., and the Illinois Soybean Checkoff Board (ISCB) for a grand two year total of $137,000. Additional support for year three and possibly year four is pending upon progress by C-FAR and the ISCB. Members of the research team are also involved with a large special research initiative (SRI) sponsored by the ISCB entitled the "Soy/Swine Nutrition Nutrition Program⁽¹⁵⁾." A total budget of $1.3 million was allocated for the first year of the project, and includes researchers from eleven institutions. The principal investigator (TAW) of this project is a team co-leader of one of the objectives of the SRI: To investigate the effects of the bioactive molecules in soybeean meal on reproduction and carcass composition. Other members of the team include Dr. Tim Stahly (co-leader), Iowa State University who is examining the effects of soy isoflavones on growth, immune function, and body composition of pigs; Dr. Walter Hurley, University of Illinois, who is examining the effects of soybean phytoestrogens on the porcine mammary gland; and Dr. Matt Wheeler, University of Illinois, who is examining the influence of isoflavones on embryonic development and embryonic gene expression. Dr. Winters is examining soy phytoestrogen effects on the ovary. Additional funding is expected over the next two years to continue this funding. All of these projects are closely related to the objective of this IPPA funded project.

Dissemination

A webpage has been set up at http://www.siu.edu/~tw3a/soyrepro.htm to update the public on the progress of this and other related projects. No scientific papers have yet resulted from the project, but Dr. Winters has presented some of the information at invited lectures at Iowa State University⁽¹⁶⁾ and the 1999 Midwest Section Animal Science Meetings⁽¹⁷⁾.

Literature cited:

1. Knight, D.C. and J. Eden. 1996. A review of the clinical effects of phytoestrogens. Obstet. Gynecol. 87:897-904,

2. Adams, N.R. 1995. Detection of the effects of phytoestrogens on sheep and cattle. J. Anim. Sci. 73:1509-1515.

3. Drane, H.M., A.E. Wrathall, D.S.P. Patterson, and C.N. Hebert. 1981. Possible oestrogenic effects of feeding soyameal to prepubertal gilts. Br. Vet. J. 137:283-288.

4. Urban, R.J., Garmey, J.D., Shupnik, M.A. and Veldhuis, J.D.: Insulin-like growth factor type I increases concentrations of messenger ribonucleic acid encoding cytochrome P450scc cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells. Endocrinology 127:2481-2488, 1990.

5. Billig, H., I. Fuerta, and A.J.W. Hsueh. 1993. Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133:2204-2212.

6. Niemann, H., and F. Elsaesser. 1987. Steroid Hormones in early pig development. In: The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation In Vitro, B.D. Bavister (Ed.) Plenum Press, New York, NY, pp. 117-132.

7. Winters, T. A., J. A. Greer, S. R. Read, M. P. Williams, A. M. Suttner, G. A. Byassee, M. L. Doran, D. A. Lightfoot, and W. J. Banz. 1997. Dietary soy protein and phytoestrogens: A histological examination of reproductive tissues in rats. Symposium on Phytoestrogen Research Methods: Chemistry, Analysis, and Biological Properties. Tuscon, AZ. September 23., Poster #24.

8. Carnevale, E.M., M. Uson, B.L. Soderstrom, J.J. Crawford, and T.A. Winters. 1998. Effects of phytoestrogens on the reproductive tract of anestrous mares. Annual Meeting of The Society for Theriogenology and the American Association of Equine Practitioners Convention, Baltimore, Md., December 6-9.

9. Suttner, A., N.A. Danilovich, W.J. Banz, and T.A. Winters. 1998. The effects of the phytoestrogen daidzein on in situ apoptosis in primary porcine granulosa cells. Biol. Reprod. 58 (Suppl.1): 7.

10. Raymer, A.M., N.A. Hoefling, W.J. Banz, and T.A. Winters. 1999. Soy isoflavones: Effects on in situ apoptosis in swine ovarian granulosa cells. Sixth World Soybean Research Conference, Global Soy Forum 99, Chicago, IL, Aug. 4-7. (Accepted)

11. Bibbo, M., W.B. Gill, A. Freidoon, R. Blough, V.S. Fang, R.L. Rosenfield, G.F.B. Schumacher, K. Sleeper, M.G. Sonek and G.L. Weid. 1977. Follow-up study of male and female offspring of DES-exposed mother. J. Obstet. Gyn. 49:1-7.

12. Drickamer, L.C., R.D. Arthur, and T.L. Rosenthal. 1997. Conception failure in swine: Importance of the sex ratio of a female's birth litter and tests of other factors. J. Anim. Sci. 75:2192-2196.

13. Smith RM, Tiesinga JJ, Shah N, Smith JA, Jarett L: Genistein inhibits insulin-stimulated glucose transport and decreases immunocytochemical labeling of GLUT4 carboxyl-terminus without affecting translocation of GLUT4 in isolated rat adipocytes. Proc Natl Acad Sci 88:6893-6897, 1991

14. Banz, W. J., M. P. Williams, J. A. Greer, S. R.. Read, G. A. Byassee, D. A. Lightfoot, T. A. Winters. 1997. The effects of soy protein and soy phytoestrogens on symptoms associated with cardiovascular disease in rats. Symposium on Phytoestrogen Research Methods: Chemistry, Analysis, and Biological Properties. Tuscon, AZ. September 23., Poster #23.

15. Sonka, S.T., R. Easter, and M. Nash. 1999. Soy Swine Nutrition Research Program Bulletin. National Soybean Research Laboratory, Univ. of Illinois. 1:1-8.

16. Winters, T.A. 1999. Soy Phytoestrogens & Animal Reproduction. Wise and Helen Boroughs Lectureship. The Nutritional Sciences Council, Iowa State University. Lecture # 148, Feb. 3.

17. Winters, T.A. 1999. Soy Phytoestrogens in Swine Production. Invited Lecture at Nonruminant Nutrition Symposium entitled, "Nutraceuticals: Nutrition or Nirvana?" 32^nd Midwestern Sectional Meeting of the America Association of Animal Science, March 15.

Summary of Relevance to Illinois Pork Industry

Our research hypothesis was that phytoestrogens in soybean meal can positively affect ovulation rate and embryonic survival in swine, thus increasing average litter size. An average increase of one pig per litter nationwide would allow us to produce 17 million more market pigs with the same number of breeding females to meet expanding world needs (i.e., export market). Preliminary data suggests that ovulation rate may be positively affected. In addition, growth results suggest that soy phytoestrogens in the diet may lead to a larger framed and leaner pig. If these results turn out to be true, they would be economically beneficial to swine and soybean producers by making pork production more efficient and by increasing the utilization and/or demand of soy products in the swine industry.