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Expression of genes involved in regulation of cell turnover during milk stasis and lactation rescue in sow mammary glands¹

P. K. Theil^*,2, R. Labouriau, K. Sejrsen^*, B. Thomsen and M. T. Sørensen^*

^* Departments of Animal Health, Welfare, and Nutrition and and Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Research Centre Foulum, Tjele, Denmark

	Abstract

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We studied the transcription of selected genes involved in the regulation of cell turnover during milk stasis and lactation rescue in individual mammary glands of sows and evaluated the timeframe for lactation rescue and the subsequent productivity of rescued glands. Suckling was prevented in two glands from each of five sows by taping them the day after farrowing for 1 or 3 d (Closed 24 h and Closed 72 h glands, respectively). After removing the tape, Closed 24 h glands were suckled until weaning. Closed 72 h glands were refused by the piglets and therefore remained unsuckled. Mammary gland biopsies were collected to quantify the transcription of genes involved in cell turnover and the percentage of proliferating and apoptotic cells. Transcriptional data were analyzed with a -distributed generalized linear mixed model. Mammary transcription of -lactalbumin was high in suckled glands the day after farrowing, indicating onset of lactation, but was downregulated after 1 d of milk stasis. The prolac-tin receptor mRNA was downregulated and IGFBP-5 mRNA was upregulated within 1 d of milk stasis. The downregulation of -lactalbumin and prolactin receptor and upregulation of IGFBP-5 mRNA noted after 1 d of milk stasis was maintained in Closed 72 h glands (those glands regressed) but reversed on d 4 and 6 of lactation in Closed 24 h glands. Mammary IGF-I mRNA was not regulated in response to milk stasis or lactation rescue. The percentage of proliferating cells in mammary glands was high prepartum (13.1%) and intermediate (7.8%) the day after farrowing. By d 6 of lactation, the percentage of proliferating cells was increased to 10.1% (P < 0.01) in glands suckled regularly but decreased to 5.9% (P < 0.05) in regressing glands (Closed 72 h glands). Glands rescued after 1 d of milk stasis had lower productivity throughout lactation than glands suckled regularly, as indicated by the BW of piglets suckling these glands (242 vs. 315 g/d, respectively; P < 0.05). In conclusion, regularly suckled glands had a greater cell proliferation, greater transcriptions of -lactalbumin and prolactin receptor genes, and less IGFBP-5 transcription compared with rescued (Closed 24 h) and regressing (Closed 72 h) glands. Glands that were not suckled for 1 d could be rescued, although their subsequent productivity was lower, whereas glands not suckled for 3 d could not be rescued.

Key Words: Cell Proliferation • Gene Expression • Lactation Rescue • Mammary Gland • Pig • Prolactin Receptor

	Introduction

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Cross-fostering is often used in swine production to ensure a milk supply to all suckling piglets. Successful cross-fostering requires that piglets are moved to the foster sow as close as possible to her farrowing date to be able to stimulate milk synthesis in formerly unsuckled glands and to be accepted by the sow (Price et al., 1994). For management reasons, cross-fostering is not always possible at the optimal time, and a solid understanding of the underlying biology of milk stasis and lactation rescue in unsuckled mammary glands is necessary to make adequate recommendations on cross-fostering.

The development of individual sow mammary glands in early lactation is closely related to suckling intensity (Kim et al., 1999). In contrast, nonsuckling induces mammary involution within a few days of milk stasis (Kim et al., 2001). This strongly suggests that mammary development during lactation is regulated at the level of the mammary gland (Hartmann et al., 1997). It also suggests that the biological effect of prolactin (PRL), which has a major effect on mammary growth in sows (Farmer, 2001), is inhibited at the local level in unsuckled glands.

Prolactin acts via binding to the PRL receptor, and changes in expression of the PRL receptor may therefore be involved in the regulation of cell turnover. An alternative control mechanism may be a decrease in mammary expression of IGF-I because overexpression of IGF-I was reported to delay mammary gland involution (Neuenschwander et al., 1996). Increased expression of IGFBP-5 also may affect mammary development because it is involved in the regulation of apoptosis (Tonner et al., 2000).

The goals of the present experiment were to study the expression of the PRL receptor and of IGF-I and IGFBP-5 in suckled and unsuckled mammary glands of sows, and to investigate mammary cell turnover and lactation productivity in relation to milk stasis and lactation rescue in glands that were not suckled for 1 or 3 d postpartum.

	Materials and Methods

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Animals
Five multiparous sows (Landrace x Yorkshire) were selected randomly from the herd at the Research Centre Foulum and kept individually in farrowing pens. They were fed twice daily in accordance with Danish recommendations (Danielsen, 1988). The diet was based (as fed) mainly on barley (40%), wheat (32%), and soybean meal (22%), and contained 187 g of CP and 15.25 MJ of ME/kg of DM. The feeding level was gradually increased within the first 10 d of lactation, until the daily supply of ME was 86 MJ. All litters were standardized to 10 piglets by removing extra littermates on d 1. Throughout lactation, sows and piglets had free access to water. Piglets were given electrolytes (Dialyte; Wittfoss, Graasten, Denmark) in a bowl from d 6 to 9 of lactation and had free access to a starter diet based mainly on barley (32%), wheat (32%), soybean meal (14%), and fish-meal (12%) on an as-fed basis (222 g of CP and 14.81 MJ of ME/kg DM) from d 14 of lactation. On d 1 to 3, sows received a prophylactic treatment for mastitis via a daily injection of 10 mL of Engemycin (100 mg/mL of oxytetracyclin; Intervet, Skovlunde, Denmark). All piglets were weighed individually, and teat order was recorded during the first suckling bout after each feeding (twice daily) during the first week of lactation and then twice daily two times per week until piglets were weaned on d 28 of lactation.

Suckling Regimens
Three of the five anterior glands on the left side of the udder of each sow were exposed to three different suckling regimens from d 1 to 4 of lactation as follows: 1) the gland was sucked throughout lactation (Suckled); 2) the gland was not sucked for 24 h beginning 24 to 36 h postpartum (Closed 24 h); or 3) the gland was not suckled for 72 h beginning 24 to 36 h postpartum (Closed 72 h). The two remaining anterior glands on the left side of the udder were taped either before farrowing or 12 to14 h postpartum to study the role of suckling on the onset of lactation (data not shown). Nonsuckling was achieved by sealing the glands with adhesive tape, which was changed every other day. Treatments were allocated randomly to teat position one to five among the five sows. The piglets were left by the sow to fend for themselves while their teat was taped; however, at least 10 glands were accessible for suckling at all times.

Biopsies and Observations
Biopsies of mammary glands were collected while the sows were held by snare restraint. Biopsies were taken from glands selected for the Suckled regimen approximately 5 d before the expected farrowing (d –5) and from all three selected glands on d 1, 2, 4, and 6 of lactation. Five minutes before the collection of biopsies, the udder was washed, disinfected with ethanol, and anesthetized locally by two splashes (a splash is equivalent to approximately 10 mg of xylocain or 0.1 mL of solution) of xylocain cutane spray (AstraZeneca A/S, Albertslund, Denmark) at the intrusion site (approximately midway between the teat and the upper line of the udder). Care was taken not to perforate blood vessels visible on the gland surface. The biopsies were taken with a Manan Pro-Mag 2.2 biopsy gun (Medical Device Technologies, Gainesville, FL) loaded with a 14-gauge needle. The needle was forced approximately 4 cm into the gland before pulling the trigger and was pointing slightly downward. A biopsy consisted of up to three shots in the same intrusion site, so that a total amount of 30 to 60 mg of gland tissue was collected. Approximately 5 to 10 mg of the first biopsy was stored in formaldehyde (4%) for histological staining. The remaining tissue was immediately frozen in liquid N₂ and stored at –80°C for later analysis of quantitative gene expression by means of real-time reverse-transcription (RT) PCR. On d 1, 2, 4, 6, 13, 20, and 27, all glands on both mammary sides were scored on a scale ranging from 1 (regressed gland) to 5 (fully developed gland). Visual scoring was done by one person for consistency. The Danish Experimental Inspectorate, Ministry of Justice, (Copenhagen, Denmark) approved the experiment.

Immunohistochemistry
Tissue was fixed overnight in 4% (vol/vol) formaldehyde, dehydrated in an ethanol series, and embedded in paraffin. Sections (4 µm) were cut on a microtome. Proliferating cells were stained with monoclonal antibody mouse/anti-human Ki-67 clone mib-1 and Dako EnVision plus system (DakoCytomation Norden A/S, Glostrup, Denmark). Apoptosis was quantified by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay by using the apoptag plus peroxidase in situ apoptosis detection kit (Chemicon Int., Temecula, CA). The manufacturer’s recommendations were followed, and at least 1,500 cells were counted from at least five random fields within the same section. Cell proliferation and apoptosis were expressed as the percentage of total counted cells that were stained by either a cell proliferation antigen (Ki-67) or with TU-NEL assay, respectively.

Real-Time RT-PCR
Approximately 10 mg of gland tissue was homogenized in 350 µL of RNeasy lysis buffer and the homogenate was diluted with 70% ethanol (1:1). The RNA was purified using the RNeasy mini kit (Qiagen, Albertslund, Denmark). Purified RNA was reverse-transcribed with oligo-dT and a Superscript II RNAse H reverse transcriptase kit (Invitrogen, Taastrup, Denmark) according to the manufacturer’s protocol. Reverse-transcribed material (1 µL) was amplified with TaqMan Universal PCR Master Mix (Applied Biosystems, Stockholm, Sweden) using primer pairs specific for each gene and signal was detected quantitatively by a gene-specific minor groove binding probe-labeled with FAM (carboxyfluorescein) fluorophore on the 5' end. Primers and probes were designed by using Primer Express Version 2.0 software (Applied Biosystems, Stockholm, Sweden), and for all genes, either a primer or the probe annealed to a splice site. ß-Actin and glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) were used as endogenous controls (housekeeping genes) and -lactalbumin as an indicator of initiation or rescue of lactation. No amplification was found in ribonuclease-free water and in samples of genomic pig DNA. For RT-PCR, 40 cycles were used at 95°C for 15 s, and 60°C for 60 s. The response was quantified as the number of PCR cycles required to reach a certain threshold, and samples were analyzed in duplicates. The oligonucleotide sequences of forward primers, minor groove binding probes, and reverse primers for the genes were as follows: -lactalbumin = 5'-acaatggcagcacagaatatgg, 5'-ctcttccagatcaataat, 5'-tcagtaaggtcatcatccaggaatt; prolactin receptor: 5'-ggctccgtttgaagaaccaa, 5'-caaggag accccgcc, 5'-gtctttcgcagctggattctg; IGF-I: 5'-gctggtggac gctcttcagt, 5'-cgtgtgcggagacag, 5'-ccgtaccctgtgggcttgt; IGFBP-5: 5'-gaccgcaagggattctacaaga, 5'-aaagcagtgcaa gccttcccgtgg, 5'-tccacgcaccagcagatg; ß-actin: 5'-tccagag gcgctcttcca, 5'-tcctgggcatggagt, 5'-cgcacttcatgatcgagtt ga; GAPDH: 5'-gtcggagtgaacggatttgg, 5'-cgcctggtcacc agggctgct, 5'-caatgtccactttgccagagttaa.

Statistical Analyses
Body weight and BW gains of the piglets suckling each selected gland were analyzed using a normal mixed model (SAS MIXED; SAS Inst., Inc., Cary, NC). The mammary cell proliferation and mammary apoptosis were analyzed using a binomial mixed model (SAS macro GLIMMIX) as described by Littell et al. (1996). The observations were dependent in all of these analyses due to the design because measurements were taken from the same sow and the same gland (or piglet) at different times. Incorporating two suitable defined random components in the referred models accounted for this correlation structure. Standard contrasts, suitably defined, for mixed models were used for comparing treatment differences. Comparison of BW gains was only performed for Suckled and Closed 24 h glands because Closed 72 h glands remained unsuckled. A Friedman test (Siegel and Castellan, 1988) was used for comparisons of gland scoring between the different treatments.

The relative gene expressions obtained from the RT-PCR determinations were tested by fitting a suitable gamma mixed model constructed on the basis of kinetics involved in RT-PCR amplifications. The model specified that for each combination of suckling regimen and lactation day (indexed by v), the numbers of PCR cycles, C_t, were related to the mean expression of the two housekeeping genes (GAPDH and ß-actin), R_v, according to the following:

[1]

where Z_s and Z_sg are (standard) normal random components applied to account for repeated measurements within sow and within gland x sow, respectively; ²_s and ²_sg are the related variance component; ₀ and ß₀ are the intercept and the slope of the regression relating the number of PCR cycles to the relative concentrations of standards; and _v is the relative expression of the gene for the combinations of suckling regimen and lactation day. The model defined in Eq. [1], when taking ₀ and ß₀ as known and assuming the response variable gamma-distributed, is a generalized linear mixed model (Fahrmeir and Tutz, 2001), which was fit using an adapted version of the SAS GLIMMIX macro (Littell et al., 1996).

	Results

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All glands from the treatments Suckled and Closed 24 h were suckled normally after the treatment period until the piglets were weaned 28 d after farrowing. In contrast, Closed 72 h glands remained unsuckled after tape removal on d 4, most likely because the glands had become dry. One piglet from each of two litters died (d 6 and 12, respectively). At the end of lactation, each piglet suckled its own active gland (i.e., three sows had 10 lactating glands, whereas the other two sows had nine active glands).

Gland and Piglet Performance
As shown in Figure 1A, the productivity of rescued (Closed 24 h) glands was decreased compared with that of Suckled glands (242 vs. 315 g/d, respectively; P < 0.05). Based on visual scoring of mammary glands, the development of Closed 24 h glands was similar to that of Suckled glands throughout lactation (Figure 1B). In contrast, Closed 72 h glands clearly regressed, and the regression was visible approximately from d 6 of lactation. The body weight of piglets using Suckled glands and Closed 24 h glands was similar before glands were taped (Table 1), as evaluated by the BW of the piglets that later on selected these particular glands. On d 4 and 6 of lactation, BW of piglets using Suckled glands was greater than that of piglets using Closed 24 h glands (P < 0.05).

View larger version (21K): [in this window] [in a new window]   Figure 1. Body weight gain of piglets suckling mammary glands exposed to a different suckling regimen beginning 24 to 36 h postpartum (Suckled, Closed 24 h, Closed 72 h; A) and mean visual score of mammary glands during lactation in response to suckling regimen (B). Piglet BW gains were recorded for d 1 to 2, 2 to 4, 4 to 6, 6 to 13, 13 to 20, and 20 to 27 (BW of piglets are given for piglets on d 1 to 6 in Table 1). Note that a single piglet suckled a Closed 72 h gland for 2 d (d 4 to 6) after tape was removed. Scoring of mammary glands was performed on an arbitrary scale ranging from 1 (fully regressed gland) to 5 (fully developed gland) and was done every other day during the first week of lactation, and then weekly until weaning (d 28). Different letters indicate significant differences (P < 0.05) between suckling regimens within a day. Dotted lines indicate periods in which Closed 24 h or Closed 72 h glands were covered by tape.

The growth and productivity of mammary glands suckled regularly was not influenced by repetitive biopsies. Except for the first 2 d postpartum, the BW gain by piglets using Suckled glands (left side of the udder) was well above 200 g/d throughout lactation. In addition, piglet BW gain supported by Suckled glands (left side), where biopsies were collected (315 g/d), was similar to that supported by glands on the right udder half (298 g/d), where no biopsies were taken (P = 0.76).

Cell Proliferation and Apoptosis
The percentage of proliferating cells decreased from 13.1% 5 d prepartum to 7.8% on d 1 postpartum. During the first week of lactation (Table 1), the percentage of proliferating cells increased to 10.1% in Suckled glands (P < 0.01) but decreased to 5.9% (P < 0.05) in glands where lactation was not rescued (Closed 72 h). Cell proliferation in Closed 24 h was comparable with that of Suckled glands on d 2 and 4, whereas cell proliferation was decreased in Closed 24 h glands by d 6 (P < 0.01). The median percentage of apoptotic cells was greatest for Closed 72 h glands, but no statistical differences were detected (Table 1).

A total of zero apoptotic cells (when counting at least 1,500 cells) was found in 40% of the samples stained by the TUNEL assay, but there was no pattern that could be explained by the suckling regimens. Statistical analysis revealed an over-dispersion of this measure (P < 0.001 for testing equality of the overdispersion parameter to Model [1]. The CV was in the range of 93 to 224%, confirming that the overdispersion was not only statistically significant but also of considerable magnitude.

Abundance of mRNA
The treatment x day interaction was significant (P < 0.001) for mRNA abundance of all genes studied. No significant differences in mRNA abundances were detected on d 1, which is due to the fact that biopsies were taken before glands were taped on d 1. The abundance of -lactalbumin (Figure 2A) was five- to sixfold greater in all glands at d 1 of lactation compared with the prepartum level (P < 0.001). The -lactalbumin mRNA abundance remained stable in Suckled glands throughout the study, except for a low abundance on d 2 (P < 0.01). The transcription pattern in Closed 24 h glands was comparable to that of Suckled glands, except on d 4, when -lactalbumin mRNA abundance was less than in Closed 24 h glands (P < 0.05). The abundance of -lactalbumin was less in Closed 72 h glands than in Suckled glands on d 2 (P < 0.05) reaching the prepartum levels at d 4, and remaining low beyond that.

View larger version (24K): [in this window] [in a new window]   Figure 2. Abundance of mRNA for -lactalbumin (A), prolactin (PRL) receptor (B), IGF-I (C), and IGFBP-5 (D) relative to prepartum levels (in arbitrary units = AU) in response to suckling regimen (Suckled, Closed 24 h, Closed 72 h) during the peripartum period. Dotted lines indicate periods where Closed 24 h or Closed 72 h glands were covered by tape. Different letters indicate a significant difference (P < 0.05) between suckling regimens within a day.

The mRNA abundance of IGF-I decreased slightly in Suckled and Closed 24 h glands with progress of lactation (P < 0.05); however, by d 4 and 6 of lactation, the abundance of IGF-I mRNA was greater in Closed 72 h glands (P < 0.05; Figure 2C) than in Suckled glands. The transcription pattern of IFGBP-5 remained stable in Suckled glands throughout the study (P > 0.21). Compared with Suckled glands, the abundance of IGFBP-5 mRNA was transiently elevated by 2.5-fold in Closed 24 h glands on d 2 (P < 0.01; Figure 2D) and elevated by 2.5- to threefold (relative to prepartum levels) in Closed 72 h glands, on d 2 through 6 (P < 0.01). The transcription of PRL receptor was negatively correlated with that of IGFBP-5 for all suckling treatments. The Pearson correlation coefficient was –0.51 (P < 0.001), –0.83 (P < 0.001), and –0.29 (P < 0.05) for Closed 72 h, Closed 24 h, and Suckled glands, respectively.

	Discussion

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The present study suggests that the expression of PRL receptor gene may be a key point in the regulation of cell turnover and possibly also mammary gland productivity during milk stasis and lactation rescue. Expression of the PRL receptor gene was increased in late pregnancy and remained high in Suckled glands throughout the first week of lactation. This finding underlines the importance of the PRL signal for proper mammary development in pregnant sows (Farmer et al., 2000) and for productivity of mammary glands in lactating sows (Farmer et al., 1998). Expression of the PRL receptor gene was less in unsuckled glands and may therefore be the initial signal for regression in response to milk stasis. Although not significantly different from controls, subsequent suckling of glands taped for 1 d partly restored the expression of the PRL receptor gene to values similar to that of Suckled glands. The numerically lower expression of the PRL receptor gene coincided with a lower productivity throughout lactation in glands not suckled for 1 d. Overall, present results indicate that the involution process in individual mammary glands involves a lower expression of the PRL receptor in response to milk stasis, whereby regression of individual glands may occur in spite of high concentrations of circulating PRL.

Mammary expression of IGF-I also could be involved in the regulation of mammary cell turnover in glands that are either suckled or not suckled. In support of this, Neuenschwander et al. (1996) found that mice overexpressing IGF-I had a delayed involution after weaning. Furthermore, IGF-I is a potent growth factor capable of stimulating terminal end bud formation and ductal morphogenesis in developing mammary glands (Kleinberg et al., 2000); however, present data showed that IGF-I expression in mammary tissue could not account for the regression of unsuckled glands or for the increased growth of suckled glands. In contrast, IGF-I was upregulated in regressing (Closed 72 h) glands on d 4 and 6, which may be a response to the apoptotic signal elicited by the increased expression of IGFBP-5.

The expression of IGFBP-5 increased within 24 h of milk stasis, indicating the induction of apoptosis (Tonner et al., 1997; 2000). Thereafter, IGFBP-5 expression was downregulated in Closed 24 h glands, coinciding with lactation rescue. In contrast, in the glands where lactation was not rescued after not being suckled for 3 d (Closed 72 h), IGFBP-5 expression remained upregulated throughout the first week of lactation. This prolonged upregulation supports the role of IGFBP-5 in mammary tissue remodeling, as proposed by Tonner et al. (2000).

Our results suggest that upregulation of IGFBP-5 brought about by milk stasis may be reversible after 24 h of no suckling, but that it is irreversible after 72 h. The reversible phase may be important for individual glands to adjust their productivity to the suckling stimulus of the piglet. This occurs during the first several days of lactation (W. L. Hurley, Univ. of Illinois, Urbana; personal communication). On the other hand, the irreversible phase signals gland regression and probably also tissue remodelling and may explain that glands reach a state where lactation cannot be rescued ("point of no return"). Hence, present data suggest that mammary expression of the PRL receptor and of IGFBP-5, but not of IGF-I, are important local factors signaling continued development in suckled glands or regression in unsuckled glands. The mechanism of PRL-stimulated growth may involve a suppression of IGFBP-5 expression. Tonner et al. (1997) found that the injection of PRL inhibited the greater IGFBP-5 protein expression induced in rat mammary glands by litter removal. In support of the inhibitory role of PRL on IGFBP-5 expression, the expressions of PRL receptor and IGFBP-5 were negatively correlated in the present study.

Cell proliferation was stimulated through d 6 of lactation in glands suckled regularly, but it was depressed in glands not suckled for either 1 or 3 d (Table 1). This could partially account for the observed differences in gland development evaluated by visual scoring. Cell proliferation rate determines, in concert with the rate of apoptosis, the net gain or loss of mammary tissue (Capuco et al., 2001) and thereby affects the capacity for milk production (Wilde et al., 1997). The percentage of apoptotic cells was greater in unsuckled (Closed 24 h and Closed 72 h) glands the day after the glands were taped, and the percentage of apoptotic cells remained high in regressing (Closed 72 h) glands. Nonetheless, the analysis yielded divergent results (CV was 93 to 224%), and therefore no differences were statistically significant. We found no apoptotic cells present in 40% of all samples, even though several of these samples originated from glands not suckled for 3 d (Closed 72 h glands), for which high levels of apoptosis could be expected based on the visual scorings and expression of IGFBP-5. This finding suggests that apoptosis may not be evenly distributed within the mammary gland, but rather that it is concentrated at local sites. If so, an average measure of apoptosis cannot be expected from immunohistochemical analysis of cells from sections that originate from one site. Altogether, the present findings suggest that mammary development of suckled glands is obtained by stimulation of cell proliferation and inhibition of apoptosis. Mammary gland regression is most likely obtained by both inhibited cell proliferation and stimulated apoptosis.

Lactation was rescued in glands that were not suckled for 24 h in all sows, but the productivity of these glands (estimated by piglet BW gain) was less than that of glands suckled throughout lactation. There are two likely explanations for this: 1) mammary development may have been arrested due to milk stasis for 1 d; or 2) the piglets suckling the rescued glands weighed less than their littermates, thereby decreasing the suckling stimulus exerted on the mammary gland (Hurley, 2001). Therefore, it cannot be concluded whether the observed decrease in lactation productivity in rescued glands was in fact due to irreversible loss of milk-producing capacity or to a lesser gland stimulation. Regardless of the cause, cross-fostering later than d 1 of lactation will increase the within-litter variation of piglet BW at weaning due to the lowered milk intake by some of the littermates during lactation. In agreement, Thorup (1998) found that piglets cross-fostered later than 1 d after the foster sow farrowed weighed 900 g less at weaning than control piglets (6.1 vs. 7.0 kg, respectively).

	Implications

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Present results suggest that cross-fostering should be done within 24 h after the foster sow has farrowed. Cross-fostering later than 24 h postpartum is possible, but the present study indicates that the piglet may not be able to fully initiate lactation at this time in a formerly unsuckled gland. Cross-fostering cannot be performed after 3 d postpartum because unsuckled glands have passed the point of no return, and piglets are no longer able to rescue lactation in these glands. Successful cross-fostering requires the acceptance of the sow of cross-fostered piglets and access to a lactating gland for each suckling piglet. The mammary prolactin receptor and insulin-like growth factor binding protein-5 seem to play a central role in regulating the development of suckled glands and the regression of unsuckled mammary glands of sows. Indeed, the lower productivity of rescued glands and the simultaneous lower expression of prolactin receptors suggest that the prolactin receptor may be a limiting factor for milk production.

	Footnotes

 
¹ The Danish Agricultural and Veterinary Research Council supported the project (Grant No. 23-02-0115). We thank W. Hurley, Univ. of Illinois, Urbana-Champaign, for fruitful discussions and for comments to the manuscript. H. Handll is thanked for help in the stable and for skillfully performing all the analyses.

² Correspondence: P.O. Box 50, DK-8830 (phone: +45 8999 1160; fax: +45 8999 1525; e-mail: Peter.Theil{at}agrsci.dk).

Received for publication February 14, 2005. Accepted for publication June 8, 2005.

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