Effects of Supplemental @-Carotene and Vitamin A on Reproduction in Swine’f2 Boon P. Chew Department of Animal Sciences, Washington State University, Pullman 99164-6320

ABSTRACT

Since its discovery some 80 yr ago, many advances have been made in understanding the many diverse roles of vitamin A (retinoids). Among these are the critical roles that vitamin A plays in regulating reproduction in both the male and female. The identification of retinol-binding proteins produced by the pig uterus and conceptus marks an exciting

event. It will be pivotal to future efforts in elucidating the mechanism by which retinoids regulate conceptus development and steroidogenesis. However, relatively little is known of the possible direct role played by its provitamin, /3-carotene, in controlling reproduction in the pig. However, future research likely will address this aspect of carotenoid function.

Key Words: Reproduction, Pigs, Vitamin A, &Carotene

J. h i m . Sci. 1993. 71:247-252

Introduction The importance of vitamin A (retinoids) in regulating vision (Dowling and Wald, 19601, growth (Zile and Cullum, 19831, reproduction (Thompson, 197.51, and health (Chew, 1987) has been documented. Vitamin A exists in three naturally occurring forms: retinol, retinoic acid, and retinal (Frickel, 1984). These retinoids differ only in their chemical oxidation state, and each form plays a somewhat unique role in regulating physiological functions. For example, retinol serves as the major transport form of vitamin A and, when esterified, serves as the storage form in the liver. Retinaldehyde is the unique chromophore for rhodopsin, the visual protein. Retinoic acid has important biological functions in maintaining cellular differentiation. However, retinoic acid is unable to support certain aspects of male and female reproduction or vision (Zile and Cullum, 1983). Preformed sources of vitamin A are found mainly in the animal kingdom, whereas plants do not contain significant amounts of vitamin A. Plant sources (and photosynthetic microorganisms) contain precursor forms of vitamin A called carotenoids. Of the > 600 carotenoids found in nature

lpresented at a symposium titled “High Levels of Supplemental Vitamins on Reproduction of‘ Swine” at the ASAS 82nd Annu. Mtg., Ames, IA. Financial support was provided by Hoffman-LaRoche, Rhone-Poulenc, and Duphar Nutrition. asupported by the Agric. Res. Sta., College of Agric. and Home Econ., Washington State Univ., Pullman, Project no. 0525. Received June 17, 1991. Accepted August 19, 1992.

(Straub, 19871, < 10% possess provitamin A activity (Olson, 1989). @-Carotene is the most biologically active provitamin A. Most animals are able to convert P-carotene to vitamin A at the lining of the intestinal wall. However, the efficiency of conversion of 6carotene to vitamin A differs with species. For example, 1 mg of @-caroteneis equivalent to 261 IU (based on a corn diet; Wellenreiter et al., 1969) to 500 IU (NRC, 1988) of vitamin A in pigs, with lower conversion efficiencies observed with higher (3-carotene intake (Wellenreiter et al., 1969). These conversion efficiencies are three to six times lower than those for poultry and rats. In addition, large species differences occur in an animal’s ability to absorb 0carotene intact. For example, carotenoids are not normally detected or are present in extremely low concentrations in the peripheral circulation of pigs (Chew et al., 1984). This is in contrast to cattle and humans, which absorb large amounts of @carotene and other carotenoids such as a-carotene, lycopene, lutein, and cryptoxanthin (Parker, 1989). Species differences in &carotene conversion to vitamin A and absorption of &carotene could be due to differences in the amount and activity of conversion enzymes, presence or absence of transport proteins, and other factors. Research on the possible specific role of 0-carotene (and other carotenoids) previously has been hampered by the assumption that its sole function in animals is t o provide vitamin A. Consequently, knowledge of the role of &carotene (and other carotenoids) on reproduction is relatively lacking compared with the knowledge we have of vitamin A. However, recent studies have shown that &carotene 247

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can modulate reproduction and other physiological functions. This review will consider the role of vitamin A and &carotene on reproduction in the pig. Where necessary, pertinent studies in other species will be incorporated.

Vitamin A and Reproduction Female Reproduction. Vitamin A is essential for the maintenance of reproduction function and fetal development (Thompson et al. 1964). The most characteristic symptoms of vitamin A deficiency in sows include the birth of weak, dead, or malformed pigs (Palludan, 1975). Vitamin A could exert these effects by influencing ovarian steroidogenesis and the uterine environment. Rats reared on a vitamin A-deficient diet have a reduced ability to secrete progesterone and 20cr-hydroxypregn-4-en-3-one into the ovarian venous blood on d 9 and 15 of pregnancy (Ganguly et al., 1971; Ganguly and Waynforth, 1971). Collagenasedispersed porcine luteal cells showed increased production of progesterone when incubated in the presence of retinol and retinoic acid (Talavera and Chew, 1988). The stimulatory effects of retinol and retinoic acid on progesterone production have also been shown with granulosa cells from rats (Bagavandoss and Midgley, 1987). The effect of retinoic acid on ovarian cell steroidogenesis is somewhat unexpected because vitamin A-deficient female rats fed retinoic acid showed decreased ability to secrete progesterone (Ganguly et al., 1971;Ganguly and Waynforth, 1971) and increased fetal resorption, the latter due to a generalized necrosis of the functional zone of the placenta. The observed difference may have been due to an insufficient concentration of retinoic acid reaching the reproduction organ in retinoic acid-supplemented animals or due to an artifact of in vitro experiments. However, the importance of retinoic acid for both male and female reproduction has been demonstrated and will be discussed later. Besides its reported effects on ovarian steroidogenesis, vitamin A could directly influence the uterine environment and the development of the embryo and fetus, or it could indirectly affect the uterine environment by affecting ovarian progesterone production. Vitamin A deficiency in pregnant sows produced structural and compositional changes in placental glycosaminoglycan (Steele and Froseth, 1980). Also, the pig uterus secretes a large amount of several proteins in response to progesterone (Roberts and Bazer, 1980, 1988). These uterine proteins are very important to the nutriture of the conceptus (Buhi et al., 1979;Roberts and Bazer, 1980).This is especially true in the pig, because the porcine trophoblast does not invade the uterine epithelium, but rather remains in superficial attachment to the uterine surface. For instance, uteroferrin, an iron-containing purple

glycoprotein (Roberts and Bazer, 1980) secreted by the pig uterine endometrium, is responsible for transporting iron to the conceptus (Buhi et al., 1979). This suggests the possible existence of other transport proteins in uterine secretions. Indeed, retinol-binding protein ( RBP) and retinoic acid-binding protein (RABP) have been found in endometrium, ovary, testis, and other tissues of mammalian species (Chytil et al., 1975;Ong and Chytil, 1975).Because vitamin A is essential for the maintenance of reproduction function and fetal development (Thompson et al., 1964), Adams et al. (1981) attempted to identify possible vitamin A-carrier proteins that can transport vitamin A from the maternal uterine endometrium to the conceptus. They reported the presence of RBP in uterine secretions from pigs in the luteal phase of the estrous cycle. Furthermore, the uterine secretion of RBP was progesterone-induced, as assessed by hormone replacement therapy using ovariectomized gilts (Adams et al., 1981). The uterine RBP identified (Adams et al., 1981) was unique to the uterus in that it differs from serum and cellular RBP in its binding affinity for retinol. These findings were supported by a subsequent study (Clawitter et al., 1990) using pseudopregnant and progesterone-treated, ovariectomized pigs. These authors reported that porcine uterine RBP is made up of at least four distinct proteins that share some sequence homology with the NHa-terminal of human serum RBP; the most acidic isoform shows the most (70% identity), whereas the most basic polypeptide shows the least (30% identity), sequence identity. Besides the pig uterine endometrium, the pig conceptus also produces RBP (Harney et al., 1990; Trout et al., 1990). Harney et al. (1990) demonstrated that RBP secretion by the pig conceptus occurs throughout the periimplantation period and that secretion occurs as early as d 10 of pregnancy, before the onset of conceptus elongation. The conceptus RBP shares very high amino acid sequence homology with human and rabbit serum RBP (Harney et al., 1990). Therefore, pig conceptus RBP is more homologous with human serum RBP than with RBP secreted into the uterine lumen of pigs. The gene encoding the RBP secreted by the early pig conceptus was recently cloned (Trout et al., 1990). These researchers isolated two apparently full-length cDNA clones (approximately 900 base pairs) for d 13 to 17 porcine conceptus RBP. They demonstrated that porcine conceptus RBP and human serum RBP share 90% nucleic acid sequence identity within the coding region and 37% identity within the 3’ non-coding region (Trout et al., 1990). Mechanism of Retinoid Action. Retinoids are lipidsoluble. Therefore, it is not surprising to find retinoids associated with water-soluble retinoid-binding proteins in plasma (Kanai et al., 1968) and in the cytoplasm (Bashor et al., 1973;Chytil and Ong, 1984) and nucleus (Giguere et al., 1987; Petkovich et al.,

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1987) of cells. Obviously a similar situation holds true in the case of the uterine milieu. Adams et al. (1981) reported that total vitamin A in uterine secretions increased in progesterone-treated pigs, thereby suggesting an increased transport of nutrients across the uterine epithelium during early pregnancy when blood progesterone is elevated. This observation, coupled with the increased secretion of RBP by both the uterine endometrium and the conceptus during the peri-implantation period, suggests increased local transport of retinoids by RBP to the developing conceptus. The mechanism for the cellular uptake of retinoids is not yet fully elucidated (Blomhoff et al., 1990). Retinoids could be taken up by target cells through a receptor-mediated mechanism, through nonspecific spontaneous transfer, or through fluidphase endocytosis (see Blomhoff et al., 1990 for review). Upon entering the cell, the retinoid is then bound to cellular retinoid-binding proteins. Cellular binding-proteins for retinol (CRBP), retinoic acid (CRABP), and retinaldehyde (CRALBP) have all been identified (for review see Blomhoff et al., 1990). In addition, the presence of nuclear retinoic acid receptors was recently reported (Giguere et al., 1987, 1990; Petkovich et al., 1987; Research News, 1990). At least three distinct forms of nuclear retinoic acid receptors (RARa, RARP, and M y ) have been identified. Another distinctly different nuclear acid retinoic acid receptor (RXRa) has recently been cloned (Mangelsdorf et al., 1990). These receptors belong to the superfamily of hormone receptor that binds to DNA. Because the cellular retinoid-binding proteins are primarily found in the cytosolic compartment, and with the recent discovery and characterization of nuclear retinoic acid receptors, two hypotheses for the role of cellular retinoid-binding proteins have been suggested. First, these cytoplasmic carrier proteins could serve to transport bound retinoid to sites of metabolism. An example of this is the cellular RBP type I1 (Ong et al., 1987), which makes bound retinol available for esterification by membrane-bound enzymes of the small intestine. Second, these cytoplasmic proteins could serve to transport bound retinoids to the nucleus, where the retinoids (retinoic acid), but not the binding protein, become bound to nuclear retinoic acid receptors. These cytoplasmic carrier proteins (especially cellular retinoic acid-binding protein) play an important role in the movement of retinoids to the nucleus, thereby regulating the concentration of the retinoids. However, it is the nuclear retinoic acid receptors that function as direct agents in gene regulation related to cell differentiation. Recent evidence indicates that the mechanism of action for retinoic acid is similar to those described for steroid hormones, thyroid hormones, and vitamin D3 (Giguere et al., 1987; Petkovich et al., 1987; Dolle et al., 1989).

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A key question is this: What is the role of retinoids in conceptus development? Retinoids may directly affect embryo development by regulating cell differentiation and proliferation (Schindler, 1986) or transcription of specific genes (Chiocca et al., 1988, 1989; Bedo et al., 19891, or possibly they act as a morphogen (Thaller and Eichele, 1987; Research News, 1990). Alternatively, retinoids may indirectly influence embryo development by regulating ovarian steroid production (Talavera and Chew, 19881, immune cell function (Chew, 19871, and interferon production (Blalock and Gifford, 1976, 1977), all of which may affect the establishment and maintenance of pregnancy. Through these combined actions, retinoids may regulate early embryonic elongation and placentation and subsequent conceptus development. Male Reproduction. The importance of vitamin A to male reproduction has been recognized for many years. Palludan ( 196 3 ) reported that histological and morphological changes of the boar testes occur before clinical signs of vitamin A deficiency appear. Earlier studies showed that spermatogenesis is arrested in vitamin A-deficient male rats (Wolbach and Howe, 1925; Mason, 1933). Spermatogenesis is a cyclic process in which Sertoli cells interact with the developing germinal cells in the seminiferous tubules. The seminiferous tubules of vitamin A-deficient male rats contain only Sertoli cells, spermatogonia, and some spermatocytes (Mitranond et al., 1979; Unni et al., 1983). Interestingly, spermatogenesis in vitamin A-deficient rats becomes stage-synchronized after vitamin A (retinol) repletion; the seminiferous epithelium was found to be almost entirely made up of only three successive stages (Morales and Griswold, 1987). Retinoic acid is unable to support reproduction. For example, retinol-deficient, retinoic acid-supplemented animals failed to reproduce and had impaired vision but otherwise showed normal growth and were healthy (Dowling and Wald, 1960). The testes of retinol-deficient, retinoic acid-supplemented animals showed atrophy of the seminiferous tubules (Thompson et al., 1964). These observations led to the belief that retinoic acid has no physiological role in the testes (Coward et al., 1969). The discovery that retinoic acid and retinol receptors are found in the rat testes (Bashor and Chytil, 1975; Ong and Chytil, 1975) led researchers to re-evaluate the possible role of retinoic acid on male reproduction. Appling and Chytil ( 198 1) provided evidence that retinoic acid indeed has a role in maintaining testosterone production in male rats. They demonstrated that retinoldepleted, retinoic acid-supplemented rats were able to maintain normal testosterone production despite atrophy of the germinal epithelium and the lack of spermatogenesis. These findings led the authors to propose that retinoic acid (in the absence of retinol) can support steroidogenesis but not spermatogenesis, whereas retinol can maintain both the steroidogenic

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(through retinoic acid) and reproductive function. Recent studies have identified two retinoic acid receptor-a mRNA (Giguere et al., 1987; Rees et al., 1989; Zelent et al., 1989) in both the Sertoli and germ cells (Kim and Griswold, 1990). The level of retinoic acid receptor-a mRNA is rather precisely regulated by retinol, suggesting that its synthesis is likely required before it can be used to modulate transcription of retinoid-inducible genes (Kim and Griswold, 1990).

0-Carotene and Reproduction In contrast to research on vitamin A and reproduction, similar studies with (?-carotene are comparatively lacking. A major reason for this is the previous assumption that 0-carotene merely serves a provitamin A role. Suggestions for a direct role of P-carotene in regulating reproductive processes in cattle have been posited (Lotthammer, 1979; Snyder and Stuart, 1981; Rakes et al., 1985). We (Michal et al., 1990) reported a lower incidence of retained placenta in dairy cows supplemented with @-carotene compared with unsupplemented cows or cows fed vitamin A. Retained placenta often leads to metritis and decreased reproductive performance (Chew et al., 1977). In contrast, others (Folman et al., 1979; Wang et al., 1982, 1988) have failed to report a beneficial effect of 0-carotene on reproduction. This discrepancy may be due to differences in the initial @-caroteneor vitamin A status of the experimental animals (Graves-Hoagland et al., 1989). Studies on the possible role of 0-carotene in regulating reproduction in white-fat animals (nonabsorbers of carotenoids) have also been reported. Rats injected with /3-carotene showed higher fetal growth rate and lower pup mortality (Chew and Archer, 1983). Similarly, crossbred gilts injected weekly with 228 mg of 0-carotene starting on the day of breeding and continuing through weaning at 3 wk postpartum had lower embryonic mortality, larger litter size, and heavier litter weight at birth and at weaning than did unsupplemented gilts (Brief and Chew, 1985). This is in general agreement with a later study (Coffey et al., 1989) in which multiparous sows were injected once at weaning with 0, 50, 100, or 200 mg of &carotene. They reported a linear increase in litter size at birth with increasing dosage of P-carotene injected. Also, the number of pigs born dead was lower in treated sows. In contrast, no treatment effect was observed with first-litter gilts treated similarly. It is unclear from the latter study whether the increased litter size at birth was due to increased ovulation rate or to decreased embryonic mortality. Because the uterine secretions are very important to the survival and development of the embryo and because these uterine secretions are progesterone-induced (Bazer, 19751, it is possible that the lower embryonic mortality observed (Brief and Chew, 1985; Coffey et al., 1989) may be due to changes in the uterine secretions and

ovarian progesterone production. Indeed, gilts injected every other day starting on the day of breeding with 16.4 mg of &carotene had quantities of uterine-specific proteins on d 15 of pregnancy higher than those of untreated pigs (Chew et al., 1982). Also, 0-carotene stimulated progesterone production by collagenasedispersed pig luteal cells by 10-fold after a 24-h incubation period (Talavera and Chew, 1988). The magnitude of response observed with P-carotene was several-fold higher than that observed with retinol or retinoic acid. In vitro, &carotene also stimulated progesterone production by bovine luteal cells; again, the stimulation profile for @-carotenewas different from that observed with the retinoids (Talavera and Chew, 1987). Further evidence for the specific role of P-carotene in regulating reproduction was provided by O’Fallon and Chew (19841, who showed that 0-carotene forms an integral component of the microsomal membrane of bovine luteal cells. Even though the studies described above show improved reproductive performance with supplemental P-carotene, it remains unclear whether 0-carotene plays a direct role in regulating certain reproductive processes or whether it merely serves as a source of vitamin A. Of course, the importance of vitamin A on reproduction is well established. More direct studies on the possible direct role of 0-carotene are needed, possibly in the identification of the putative carotenoid-binding proteins. Studies on the possible role of @-carotene on male reproduction are lacking. Mechanism of Carotenoid Action. The mechanism by which &carotene exerts its action is now known. 0Carotene could serve as an antioxidant against lipid peroxidation (Burton and Ingold, 1984), thereby protecting the uterine and ovarian steroidogenic cells from oxidative damage. In addition, 0-carotene could regulate nuclear events in target tissues. O’Fallon and Chew (1984) previously demonstrated that 0-carotene in bovine corpus luteum was covalently bound to the microsomal fraction. Also, pigs injected with 0-carotene take up 0-carotene into the subcellular components of their blood lymphocytes (Chew et al., 1990a,b), suggesting the existence of carotenoid carrier proteins.

Implications The importance of vitamin A in regulating reproduction in both male and female animals is well established. Whether supplementation of vitamin A in amounts larger than normally recommended will improve reproduction is presently unclear. @Carotene supplementation seems to improve reproductive performance in pigs by influencing ovarian steroidogenesis or changing the uterine milieu. However, it is presently unclear whether &carotene exerts this action directly or serves a provitamin A function. If 0carotene plays a direct role, then 0-carotene sup-

VITAMIN A AND REPRODUCTION IN SWINE

plementation is important because swine diets are generally low or devoid of @-carotene.The route of administration of &carotene will need to be considered because the pig absorbs little or no @-carotene.

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