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Biology of Reproduction 65, 921-927 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Effects of Breed, Parity, and Folic Acid Supplement on the Expression of Leptin and Its Receptors' Genes in Embryonic and Endometrial Tissues from Pigs at Day 25 of Gestation1

Frédéric Guaya, Marie-France Palinb, J. Jacques Matteb, and Jean-Paul Laforesta

a Department of Animal Science and Centre de Recherche en Biologie de la Reproduction, Sainte-Foy, Québec, Canada G1K 7P4 b Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Lennoxville, Québec, Canada J1M 1Z3

ABSTRACT

Recent evidence has pointed toward a possible role of leptin (Lep) and its receptor (Lepr) in early gestation materno-fetal cross-talk. However, in gestating sows, exhaustive characterization of leptin mRNA expression in backfat and leptin-receptor mRNA expression in endometrial and embryonic tissues is still pending. The objectives of this study were to characterize the Lep, Lepr, and long Lepr-L isoform mRNA expression according to the breed and parity of gestating sows or to specific folic acid (B9) + glycine dietary treatments. To this end, nulliparous (GT) and multiparous occidental Yorkshire-Landrace (YL) sows as well as multiparous Chinese Meishan-Landrace (ML) sows were used. These sows were randomly assigned to two different dietary treatments: 0 or 15 ppm of B9 + 0.6% glycine, given from the estrous preceding mating until slaughter on Day 25 of gestation. Jugular blood samples were collected at mating and on Day 25 of gestation and assayed for circulating leptin concentrations. Expression levels of Lep in backfat and of Lepr and Lepr-L in endometrial and embryonic tissues were performed using semiquantitative reverse transcription-polymerase chain reaction. Results demonstrated that on Day 25 of pregnancy, the ML sows showed higher concentrations of circulating leptin along with higher backfat thickness and higher expression of Lep in backfat tissue. Moreover, in embryonic tissues, the mRNA expression levels of Lepr and Lepr-L genes were higher in ML than in YL sows. Parity effects were observed for mRNA expression of Lepr in both endometrial and embryonic tissues, whereas mRNA levels were higher in YL than in GT sows. In addition, embryonic Lepr-L mRNA levels were higher in GT than in YL sows, and B9 + glycine dietary supplement decreased the mRNA expression levels of Lep in backfat and of Lepr in embryonic tissues. These decreases were independent of breed or parity of the sows. The effect of B9 + glycine on Lepr-L mRNA expression levels was only seen in YL sows, whereas the treatment lowered Lepr-L expression levels in both endometrial and embryonic tissues. These results indicate that leptin and its receptor may play a role during early stages of development of the pig embryo-fetus, and that these roles could be modulated according to the breed and parity of the sows. Moreover, the effects of B9 + glycine on expression levels of embryonic and endometrial Lepr-L mRNA in YL sows may explain the previously reported effects of B9 on embryo survival rate and litter size observed in occidental multiparous sows.

conceptus, implantation/early, leptin

INTRODUCTION

Leptin, the product of the obese (Lep) gene, is a 16-kDa protein that is mainly produced by adipocytes. The Lep gene was first identified in Lepob/ob mice that showed a morbid obesity, which resulted in a sterile adult with more than 50% body fat [1]. It was later demonstrated that leptin has an effect on the general metabolic rate and activity levels in decreasing feed intake [2, 3].

Since then, an increasing number of scientific papers have reported specific roles of leptin in the control of reproductive functions [4]. For example, it was previously reported that injection of recombinant leptin to Lepob/ob mice restored gonadotropin secretions, secondary sex organ weight and function, and fertility [5, 6]. It is now generally accepted that leptin acts on reproductive functions through its effect on the hypothalamic-pituitary axis [7].

Recent evidence suggests new key roles for leptin during pregnancy. For example, it was reported in humans and rats that the circulating maternal leptin concentration increases during pregnancy and then falls to less than prepregnancy concentrations around parturition [8, 9]. The localization of leptin in human placenta, amnion, amniotic fluid, chorion, and chorionic villi [10] as well as the presence of leptin and its receptor in the trophoectodermal layer of the pig [11] suggest that leptin may have a direct role in maternofetal cross-talk. Furthermore, the fact that leptin is more abundant in placenta supplying fetuses of normal growth than in those from growth-retarded fetuses has raised the possibility that leptin may be involved in growth and development of the fetus in both humans and pigs [12, 13].

In pigs, the Chinese Meishan breed is peculiar due to its high percentage of body fat, slower growth rate, and early puberty as compared to the occidental breeds [14, 15]. Meishan sows are also known for their larger litter size, with reduced embryo and fetal mortality rates [16, 17]. At the moment, cross-breeding between Meishan and occidental breeds is used to develop a prolific Sino-Occidental line for commercial herds. Although leptin-receptor mRNA expression was reported in multiple fetal and adult tissues of pigs [18, 19], to our knowledge no information is available regarding mRNA expression of leptin receptor in the embryomaternal tissues of Chinese and occidental sows during early pregnancy.

The objectives of the present study were to characterize the expression levels of swine Lep mRNA in adipose tissue as well as mRNA expression levels of the total (Lepr) and long forms (Lepr-L) of leptin receptors in endometrial and embryonic tissues on Day 25 of gestation. Possible associations of leptin and leptin-receptor expression levels with genotype and parity were evaluated in gestating sows (Day 25 of gestation), and their relation with leptin metabolism and reproductive parameters are discussed. Because folic acid (B9) increases litter size by 10% and this increase is associated with decreased embryonic mortality during early gestation [20, 21], the potential effect of a dietary supplement of B9 on leptin and its receptor was evaluated. Glycine is the most abundant amino acid in uterine secretions and allantoic fluid. The methyl group of glycine react with B9 to produce an active methyl donor needed for DNA synthesis and for remethylation of homocysteine [22–24]. Therefore, glycine was given along with B9.

MATERIALS AND METHODS

Animals and Treatments

Twenty-seven Meishan-Landrace sows (ML; 2–3 parities, LSmeans ± SEM of 193 ± 3 kg), 27 occidental Yorkshire-Landrace sows (YL; 4–5 parities, 212 ± 2 kg), and 25 nulliparous occidental Yorkshire-Landrace sows (GT; 124 ± 2 kg), which were kindly provided by Genetiporc, Inc. (St-Bernard, PQ, Canada), were used for this study. Sows in the ML and YL groups were transported to the research center a few days after the weaning of their previous litter. The GT sows were not cycling on arrival. On arrival and for at least 2 wk, all sows received 2.5 kg daily of a basal diet (50% [w/w] corn, 20% barley, 20% wheat bran, and 5% soybean meal). Heat detection was performed twice a day, between 0800 and 0900 h and between 1600 and 1700 h, by introducing a boar into the pen. On their first estrus (Day -21), sows were allocated to the following two treatments: basal diet (0 ppm), or basal diet with 15 ppm of B9 and 0.6% (w/w) glycine (15 ppm + glycine). On the second estrus, sows were inseminated twice with commercial semen (pooled semen from three Duroc boars of proven fertility; CIPQ, Inc., St-Lambert, PQ, Canada) 12 and 24 h after estrus detection. The first day of second estrus detection was considered to be Day 0 for the experiment. Dietary treatments were given until slaughter on Day 25 of pregnancy.

Samples and Measurements

Blood samples were collected at mating (Day 0) and on Day 25 of gestation to determine the serum concentration of leptin. Blood samples were centrifuged at 1800 x g for 15 min, and serum was frozen at -25°C until analysis. Body weight was determined at the first estrus (Day -21) and on Days 0 and 25. Backfat thickness was evaluated on Days -21 and 25. The backfat thickness was evaluated by an ultrasonic detector (Scanmatic SM-1; Medimatic, Hellerup, Denmark) between the third- and fourth-to-last ribs.

On Day 25, sows were slaughtered according to the recommended code of practice [25]. The reproductive tract was collected and immediately transported to the laboratory; a backfat sample was also collected and frozen immediately in liquid nitrogen. After assessing the reproductive status, only pregnant uteri were kept, and the uterine horns were dissected from the mesometrium. The ovaries, oviducts, and cervix were also removed. Embryos were localized by external palpation along each horn, and a 10-ml sample of allantoic liquid from each embryo was collected using a Vacutainer system (Becton-Dickinson & Co., Franklin Lakes, NJ). Samples of allantoic liquid from all the conceptuses of the same sow were pooled and referred to as allantoic fluid. An aliquot of that fluid was kept frozen for further analysis. Each uterine horn was then opened longitudinally along the antimesometrial aspect. The allantoic fluid from the whole litter was recovered, and the total volume was recorded. Strips of endometrium were collected from the antimesometrial and mesometrial aspects of the horns at six different sites of attachment, which were chosen at random. These strips were frozen in liquid nitrogen immediately after collection. All embryos were collected, counted, separated from the amniotic membrane, pooled, and weighed. They were then immediately homogenized with a polytron homogenizer (Kinematica, Lucerne, Switzerland) and frozen in liquid nitrogen. This embryonic preparation is referred to as embryonic homogenate. The allantochorionic membranes were collected from six different conceptuses, pooled, and frozen in liquid nitrogen. Ovaries were dissected to count the number of corpora lutea (CL). The whole procedure was completed within 35 min from slaughter.

Semiquantitative Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted from backfat and embryonic and endometrial tissues using the Trizol reagent (Gibco-BRL, Bethesda, MD) according to the manufacturer's instructions. The extracted RNA was dissolved in 50 µl of water and quantified spectrophotometrically at 260 nm. For each tissue sample, an RNA aliquot was electrophoresed in a 1% (w/v) agarose gel to verify its integrity. The cDNA was generated by a Superscript preamplification system (Gibco-BRL, Burlington, ON, Canada) according to the manufacturer's instructions. The primers and conditions used for polymerase chain reaction (PCR) amplification are shown in Table 1. For each gene, the 100-µl PCR reaction contained 200 µM dNTPs (2'deoxynucleotide 5'triphosphate), 1.5 mM MgCl2, and 2.5 U of Taq polymerase in 1x Taq polymerase buffer (Amersham Pharmacia Biotech, Baie d'Urfée, PQ, Canada). For each gene, the PCR profiles consisted of an initial denaturing step at 94°C for 2 min, an appropriate number of cycles of denaturing at 94°C for 1 min, annealing at the corresponding temperature (Table 1) for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR-amplified products were run on a 1.5% (w/v) agarose gel and stained with ethidium bromide. Pictures of the resulting gels were taken on Polaroid films (#55) (Polaroid Co., Cambridge, MA). The films were then scanned using a densitometer (Bio-Rad Imaging Densitometer Model GS-670; Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). The relative densities of transcripts were expressed as arbitrary optical units. A ratio of the optical density of leptin (Lep), its receptor (Lepr), and long isoform (Lepr-L) transcripts standardized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was calculated before statistical analyses were performed to correct for possible differences in gel loading. A control amplification of Lep, Lepr, and Lepr-L mRNA that corresponded to a single animal was also carried out on each gel to correct for possible migration or staining variations.


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  		TABLE 1. Primers and conditions used for PCR of Lep, Lepr, Lepr-L, and GAPDH genes.a

Leptin Concentrations in Serum and Allantoic Liquid

Leptin was measured in serum and allantoic liquid using a competitive radioimmunoassay (RIA) called Multi-Species Leptin RIA kit (Linco Research, St. Louis, MO). Modifications to the recommended protocol were performed as described by Mao et al. [26]. Modifications included doubling of the original sample and assay volumes and use of 0.1% egg white buffer (0.1% [w/v] egg white in PBS [0.02 M]/Tween 20 [0.05% (v/v)]/EDTA [0.025 M, pH 7.4]) for the buffer step and diluting control plasma. The intra- and interassay coefficients of variation were 6.9% and 2.9%, respectively.

Statistical Analyses

Data were analyzed using the mixed procedure of the SAS Institute, Inc. [27] according to a 3 x 2 factorial arrangement in a random design with type of sow (YL, GT, or ML) and B9 supplement (0 ppm or 15 ppm + glycine) as the two main independent factors. For serum leptin, the time of pregnancy (Days 0 and 25) was added to the model as a third factor and was analyzed using the repeated option of the mixed procedure with the autoregressive option; residual was used as the error term. For expression of endometrial and embryonic Lepr-L gene, logarithmic transformation was done to normalize experimental errors. Statistical analyses were considered to be significant when P < 0.05. Pearson correlation coefficients were determined using the correlation procedure of the SAS Institute, Inc. [27].

RESULTS

Parity and Breed Effects

The litter size and number of CL were higher in YL as compared to GT sows (P < 0.01). Although ML sows had 9% more embryos and numerically higher embryo survival rates than YL sows, these differences were not statistically significant (Table 2).


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  		TABLE 2. Effect of parity, breed, and supplementation of B9 + glycine on measures of reproductive performance on Day 25 of pregnancya

At slaughter, backfat thickness was greater in ML (27.5 ± 0.9 mm) than in YL (17.4 ± 1.1 mm) sows (P < 0.01), and YL sows had a higher backfat thickness than GT sows (11.7 ± 0.9 mm; P < 0.01). Body weight gain during gestation increased more markedly in ML (+13.8 kg/day) and GT (+14.6 kg/day) sows than in YL (+9.4 kg/day) sows (breed x time, P < 0.05; parity x time, P < 0.01).

During early pregnancy, concentrations of serum leptin were higher in ML (3.44 ± 0.26 ng/ml) than in YL (2.24 ± 0.13 ng/ml) sows (P < 0.01). The GT sows had concentrations of circulating leptin (2.33 ± 0.13 ng/ml) similar to those of YL sows. Concentrations of serum leptin increased more markedly during gestation in ML (+0.73 ng/ml) and GT (+0.47 ng/ml) sows than in YL (+0.08 ng/ml) sows (breed x time, P < 0.01; parity x time, P < 0.05).

Concentrations of serum leptin on Day 25 were associated with backfat thickness in ML (r = 0.61, P < 0.01) and YL (r = 0.55, P < 0.01) sows but not in GT sows (data not shown). However, the mRNA expression of Lep in backfat tissue was not correlated with concentrations of serum leptin and backfat thickness at Day 25 for all groups of sows (data not shown).

In backfat, the relative expression levels of Lep gene were higher in ML than in YL sows, but YL and GT sows had similar expression levels (Fig. 1).



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  		FIG. 1. Effect of parity, breed, and supplementation of B9 + glycine on mRNA expression of Lep in backfat tissue on Day 25 of pregnancy in multiparous Meishan-Landrace (ML), nulliparous Yorkshire-Landrace (GT), and multiparous Yorkshire-Landrace (YL) sows. Expression levels of Lep were determined by reverse transcript-PCR. Treatment groups were 0 ppm (control) and 15 ppm of B9 + 0.6% glycine. Data are presented as least-square means ± SEM. Breed effect, P < 0.01; B9 + glycine effect, P < 0.01

In the endometrium, Lepr mRNA expression was lower in GT than in YL sows (P < 0.01), but ML and YL sows had similar levels of Lepr mRNA expression (Fig. 2A). The parity or breed of sows had no effect on expression levels of endometrial Lepr-L gene (Fig. 2B). The number of CL was strongly correlated with the mRNA expression of endometrial Lepr in ML, GT, and YL sows (ML sows: r = 0.98, P < 0.01; GT sows: r = 0.67, P < 0.01; YL sows: r = 0.97, P < 0.01) (Fig. 3).



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  		FIG. 2. Effect of parity, breed, and supplementation of B9 + glycine on mRNA expression of Lepr (A) and Lepr-L (B) in endometrial tissue on Day 25 of pregnancy in multiparous Meishan-Landrace (ML), nulliparous Yorkshire-Landrace (GT), and multiparous Yorkshire-Landrace (YL) sows. Expression levels of Lepr and Lepr-L were determined by reverse transcription-PCR. Treatment groups were 0 ppm (control) and 15 ppm of B9 + 0.6% glycine. Data are presented as least-square means ± SEM. A) Parity effect, P < 0.01. B) Breed x B9, P < 0.01; parity x B9, P < 0.01



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  		FIG. 3. Number of CL in relation to mRNA expression of endometrial Lepr on Day 25 of gestation. A) Multiparous Meishan-Landrace sows (ML; r = 0.98, P < 0.01). B) Nulliparous Yorkshire-Landrace sows (GT; r = 0.67, P < 0.01). C) Multiparous Yorkshire-Landrace sows (YL; r = 0.97, P < 0.01). Each dot corresponds to a single pig

In embryonic tissue, ML sows had higher mRNA expression of Lepr and Lepr-L genes than YL sows (P < 0.01), and YL had higher expression levels of Lepr than GT sows (P < 0.05). However, GT sows showed higher mRNA expression of Lepr-L than YL sows (P < 0.01) (Fig. 4, A and B).



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  		FIG. 4. Effect of parity, breed, and supplementation of B9 + glycine on relative mRNA expression of Lepr (A) and Lepr-L (B) in embryonic tissue on Day 25 of pregnancy in multiparous Meishan-Landrace (ML), nulliparous Yorkshire-Landrace (GT), and multiparous Yorkshire-Landrace (YL) sows. Expression levels of Lepr and Lepr-L were determined by reverse transcription-PCR. Treatment groups were 0 ppm (control) and 15 ppm of B9 + 0.6% glycine. Data are presented as least-square means ± SEM. A) Breed effect, P < 0.01; parity effect, P < 0.05; B9 + glycine effect, P < 0.01. B) Breed effect, P < 0.01; parity effect, P < 0.01; breed x B9, P < 0.01

The mRNA expression of Lep, Lepr, and Lepr-L in the allantochorionic membrane was barely detectable. The method used in the present experiment did not allow for quantitative evaluation regarding the expression of these genes in this specific tissue (data not shown). Furthermore, the concentration of allantoic leptin could not be estimated, because it was less than the detection limits of the RIA kit (<0.33 ng/ml).

B9 + Glycine Effects

The B9 + glycine treatment had no effect on reproductive parameters, backfat thickness, body weight at Day 25 of gestation, and concentration of serum leptin for the three groups of sows. In backfat tissue, expression levels of Lep gene were decreased by B9 + glycine supplement (P < 0.01) (Fig. 1). In the endometrium, the supplement of B9 + glycine decreased the expression of Lepr-L gene in YL sows but not in ML (breed x B9 + glycine, P < 0.01) and GT sows (parity x B9 + glycine, P < 0.01) (Fig. 2B). However, the supplement of B9 + glycine had no effect on expression levels of the endometrial Lepr gene (Fig. 2A). Independent of parity or breed, the mRNA expression of Lepr was lower in embryonic tissues from sows fed the B9 + glycine supplement (P < 0.01) (Fig. 4A). However, the B9 + glycine supplement decreased the mRNA expression of Lepr-L gene in the embryos of YL but not ML sows (breed x B9 + glycine, P < 0.01) (Fig. 4B).

DISCUSSION

The Meishan breed is recognized for slower growth and higher backfat thickness compared to occidental breeds [14]. In the present experiment, ML sows, although a cross-breed between Meishan and Landrace, had a greater backfat thickness than YL sows, along with higher expression levels of Lep gene in backfat tissue and higher concentrations of serum leptin.

Such metabolic differences suggest that Meishan sows have developed an impaired metabolic utilization of leptin (i.e., high levels of fat tissue with high concentrations of leptin), as was observed in fat line pigs [28], or that selection of occidental breeds for leanness has modified sensitivity to leptin. Hormonal factors would be involved in this modified sensitivity. Indeed, the high concentrations of serum cortisol in the Meishan breed [29] would stimulate leptin secretion as well as mRNA expression of Lep gene in these animals [30].

Moreover, insulin-like growth factor I (IGF-I), which decreases the expression of Lep [31], is lower in serum of Meishan than occidental boars [32], whereas Farmer et al. [33] reported similar IGF-I values between Upton-Meishan and Large White sows during mid and late gestation. Estrogen may also be involved in this genotype effect. For example, it was reported that estradiol injection can stimulate the secretion of leptin and increase the expression levels of Lep gene in adipose tissues [34, 35]. On the other hand, some in vitro and in vivo studies have noted a lack of effect of estrogen on adipose tissue leptin secretion [36–38]. In Meishan sows, Hunter et al. [39] found higher concentrations of estradiol during the periovulatory period. However, Farmer et al. [33] reported an absence of genetic effect on estradiol values between Large White and Upton-Meishan sows during mid and late gestation. Overall hormonal results suggest that cortisol seems to be the principal hormonal factor associated with higher concentrations of serum leptin in Meishan-Landrace genotype. Finally, we cannot rule out the role of other factors, such as leptin-binding proteins, that might decrease the metabolic availability of leptin in target tissues and create leptin resistance [40].

In rodents and humans, the gestational state induces hyperleptinemia at and after midpregnancy [8, 9]. It has been suggested that this phenomenon might be associated with increased adipose tissue mass, increased secretion of leptin per adipocyte, or increased secretion of leptin from nonadipose tissues [41]. In the present experiment, the parallel observed between increased body weight and increased serum leptin concentrations, as well as the lack of allantochorionic (diffuse placenta) expression of Lep gene and the undetectable levels of leptin in the allantoic fluid, point toward adipose tissues as the major source of circulating leptin observed during early pregnancy in pigs. However, Dyer et al. [42] observed a significant expression of Lep gene in pig placental tissue at Day 75 of gestation, suggesting a possible role of placental leptin in gestating hyperleptinemia at and after midpregnancy as observed in humans and rodents [41].

It is generally accepted that the prolificacy of Meishan sows is higher than occidental sows due to higher embryo survival rates [16, 17]. In the present experiment, the increase of 1.4 fetuses in ML sows compared to YL sows, despite similar numbers of CL, was associated with a significant increase of Lepr and Lepr-L mRNA expression in embryonic tissues of ML sows. Similar results were also reported in pure-bred, gestating Meishan sows (Day 75), in which expression levels of Lepr in placental tissue were higher than those in Large White sows [42]. Such differences in Lepr and Lepr-L expression levels between Meishan and occidental breeds in placental-embryonic tissues might be associated with the different uterine environments of these breeds. These uterine differences are characterized by dissimilar concentrations and distributions of growth factors such as IGF-I and epidermal growth factor and of steroid hormones such as estradiol-17ß [43–45].

Although the role of Lep and Lepr in embryonic development of mammals is not well understood, possible roles for leptin have been proposed, including the regulation of conceptus growth and development, fetoplacental angiogenesis, and embryonic hematopoiesis [41]. Furthermore, Lepr-L acts through the STAT3 protein [46], which has a key role in murine early embryonic development [47]. However, leptin itself is not essential for embryonic and fetal development in the mouse, because Lepob/ob mice can have complete gestations [48]. Nevertheless, the present study suggests that both Lep and Lepr-L play a role in early stages of development, possibly in regulation of embryonic and placental growth [41], and that these roles could be modulated according to the genotype and parity of sows.

In the present experiment, an important correlation was found for all groups of sows between the number of CL and Lepr mRNA expression levels in endometrial tissues. Progesterone is a key hormone in the control of endometrial secretions [49, 50]. Moreover, a correlation between the number of CL and the concentrations of serum progesterone on Day 16 of the estrous cycle has been reported [51]. However, in the present study, concentrations of serum progesterone on Days 16 and 25 were not correlated with the number of CL or endometrial expression of Lepr for all groups of sows (data not shown). Therefore, other yet-unknown factors of ovarian origin could be important elements in the control of endometrial Lepr mRNA expression, which could modulate the endometrial capacity to react toward leptin [8].

Results from the present study have shown that the effects of B9 + glycine supplements on measures of reproductive performance were not significant. However, other studies have found increases in litter size and embryonic survival in occidental multiparous sows following supplementation with B9 alone [20, 21]; the present numeric increase was 1.1 fetus. Independent of breed or parity, we found that B9 + glycine treatment decreased expression levels of Lep gene in backfat tissue as well as embryonic expression of Lepr. However, for endometrial and embryonic expression levels of Lepr-L, which is the active form of leptin receptor [52], the decrease was only present in YL sows. Embryonic and endometrial expression of Lepr-L may be a key element of the differential embryonic development following supplementation with B9 in multiparous occidental sows. The effect of B9 + glycine on the expression levels of Lepr and Lepr-L in embryonic and endometrial tissues might be due to the role of B9 in the activity of S-adenosylmethionine (SAM), a molecule involved in the methylation of several other molecules, including DNA. The pattern of methylation of DNA is inheritable as well as both tissue and species specific. Moreover, methylation is a critical process for gene expression [53]. The action of B9 on SAM is dependent on the availability of other nutrients, such as vitamin B12, methionine, choline, and glycine [54]. Therefore, different nutrient statuses for GT, ML, and YL sows might explain the observed differences in the mRNA expression of endometrial and embryonic Lepr-L following supplementation with B9 + glycine. However, considering that we found no significant results for litter size, we cannot conclude that a direct link exists between the positive effects of B9 and the leptin-Lepr-L system.

In conclusion, leptin and its receptors may have an effective role in growth and development of the embryo and fetus, and this role could be modulated according to the genotype and parity of sows. The expression levels of endometrial Lepr on Day 25 of gestation may be regulated by ovarian or luteal, yet-undetermined factors. Furthermore, the effect of B9 + glycine treatment on expression levels of embryonic and endometrial Lepr-L mRNA in YL sows may, in part, be responsible for the observed effects of B9 on embryo survival rate and litter size previously reported in occidental multiparous sows.

ACKNOWLEDGMENTS

The authors are grateful to M. Guillette, A. Giguère, and D. Beaudry for technical assistance; to J. Boudreau, E. Bérubé, M. Turcotte, C. Mayrand, and F. Phaneuf for animal care; and to L. Lessard for manuscript revision.

FOOTNOTES

First decision: 8 December 2000.

1 F.G. is supported by NSERC Fellowship. This work was partially subsidized by Genetiporc, Inc. (St-Bernard, PQ, Canada), Hoffmann-LaRoche (Basel, Switzerland, and Mississauga, ON, Canada), and Agriculture and Agri-Food Canada. Lennoxville Dairy and Swine R&D Centre contribution 696. Back

2 Correspondence: Marie-France Palin, Dairy and Swine Research and Development Centre, 2000 Route 108 CP 90, Lennoxville, PQ, Canada J1M 1Z3. FAX: 819 564 5507; palinmf{at}em.agr.ca Back

Accepted: April 30, 2001.

Received: November 13, 2000.

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