Review: Mammary gland development in swine: embryo to early lactation.

Milk production by the sow is a major factor limiting the growth and survival of her litter. Understanding the process of morphogenesis of the sow's mammary gland and the factors that regulate mammary development are important for designing successful management tools that may enhance milk production. Primordia of the mammary glands are first observable in the porcine embryo at approximately 23 days of gestation. The glands then progress through a series of morphologically distinct developmental stages such that, at birth, each mammary gland is composed of the teat, an organized fat pad and two separate lactiferous ducts each with a few ducts branching into the fat pad. The glands continue to grow slowly until about 90 days of age when the rate of growth increases significantly. The increased rate of mammary gland growth coincides with the appearance of large ovarian follicles and an increase in circulating estrogen. After puberty, the continued growth of the gland and elongation and branching of the duct system into the fat pad takes place in response to the elevated levels of estrogen occurring as part of the estrous cycles. After conception, parenchymal mass of each gland increases slowly during early pregnancy and then grows increasingly rapidly during the final trimester. This growth is in response to estrogen, progesterone, prolactin and relaxin. Lobuloalveolar development occurs primarily during late pregnancy. By parturition, the fat pad of the mammary gland has been replaced by colostrum-secreting epithelial cells that line the lumen of the alveoli, lobules and small ducts. All mammary glands develop during pregnancy, however, the extent of development is dependent on the location of the mammary gland on the sow's underline. The mammary glands undergo significant functional differentiation immediately before and after farrowing with the formation of colostrum and the transition through the stages of lactogenesis. Further growth of the glands during lactation is stimulated by milk removal. Individual glands may grow or transiently regress in response to the intensity of suckling during the initial days postpartum. Attempts to enhance milk production by manipulation of mammary development at stages before lactation generally have met with limited success. A more in depth understanding of the processes regulating porcine mammary gland morphogenesis at all stages of development is needed to make further progress.

Mechanistic model to predict colostrum intake based on deuterium oxide dilution technique data and impact of gestation and prefarrowing diets on piglet intake and sow yield of colostrum.

The aims of the present study were to quantify colostrum intake (CI) of piglets using the D2O dilution technique, to develop a mechanistic model to predict CI, to compare these data with CI predicted by a previous empirical predictive model developed for bottle-fed piglets, and to study how composition of diets fed to gestating sows affected piglet CI, sow colostrum yield (CY), and colostrum composition. In total, 240 piglets from 40 litters were enriched with D2O. The CI measured by D2O from birth until 24 h after the birth of first-born piglet was on average 443 g (SD 151). Based on measured CI, a mechanistic model to predict CI was developed using piglet characteristics (24-h weight gain [WG; g], BW at birth [BWB; kg], and duration of CI [D; min]: CI, g=-106+2.26 WG+200 BWB+0.111 D-1,414 WG/D+0.0182 WG/BWB (R2=0.944). This model was used to predict the CI for all colostrum suckling piglets within the 40 litters (n=500, mean=437 g, SD=153 g) and was compared with the CI predicted by a previous empirical predictive model (mean=305 g, SD=140 g). The previous empirical model underestimated the CI by 30% compared with that obtained by the new mechanistic model. The sows were fed 1 of 4 gestation diets (n=10 per diet) based on different fiber sources (low fiber [17%] or potato pulp, pectin residue, or sugarbeet pulp [32 to 40%]) from mating until d 108 of gestation. From d 108 of gestation until parturition, sows were fed 1 of 5 prefarrowing diets (n=8 per diet) varying in supplemented fat (3% animal fat, 8% coconut oil, 8% sunflower oil, 8% fish oil, or 4% fish oil+4% octanoic acid). Sows fed diets with pectin residue or sugarbeet pulp during gestation produced colostrum with lower protein, fat, DM, and energy concentrations and higher lactose concentrations, and their piglets had greater CI as compared with sows fed potato pulp or the low-fiber diet (P<0.05), and sows fed pectin residue had a greater CY than potato pulp-fed sows (P<0.05). Prefarrowing diets affected neither CI nor CY, but the prefarrowing diet with coconut oil decreased lactose and increased DM concentrations of colostrum compared with other prefarrowing diets (P<0.05). In conclusion, the new mechanistic predictive model for CI suggests that the previous empirical predictive model underestimates CI of sow-reared piglets by 30%. It was also concluded that nutrition of sows during gestation affected CY and colostrum composition.

Level of nutrient intake affects mammary gland gene expression profiles in preweaned Holstein heifers.

Bovine mammary parenchyma (PAR) and fat pad (MFP) development are responsive to preweaning level of nutrient intake. We studied transcriptome alterations in PAR and MFP from Holstein heifer calves (n=6/treatment) fed different nutrient intakes from birth to ca. 65 d age. Conventional nutrient intake received 441 g of dry matter (DM)/d of a control milk replacer (MR) [CON; 20% crude protein (CP), 20% fat, DM basis]. Calves in the accelerated nutrition groups received 951 g/d of high-protein/low-fat MR (HPLF; 28% CP, 20% fat, DM basis), 951 g/d of high-protein/high-fat MR (HPHF; 28% CP, 28% fat, DM basis), or 1,431 g/d of HPHF (HPHF+) MR. Out of 13,000 genes evaluated, over 1,500 differentially expressed genes (DEG) were affected (false discovery rate <0.10) by level of nutrient intake in PAR or MFP. Feeding HPLF versus CON resulted in the most dramatic changes in gene expression, with 278 and 588 DEG having ≥1.5-fold change in PAR and MFP. In PAR, the most-altered molecular functions were associated with metabolism of the cell (molecular transport and lipid metabolism) with most of the genes downregulated in HPLF versus CON. In MFP, DEG also were primarily associated with metabolism but changes also occurred in genes linked to cell morphology, cell-to-cell signaling, and immune response. Compared with CON, feeding HPHF or HPHF+ did not result in substantial additional effects on DEG beyond those observed with HPLF. The pentose phosphate, mitochondrial dysfunction, and ubiquinone biosynthesis pathways were among the most enriched due to HPLF versus CON in PAR and were inhibited, whereas glycosphingolipid biosynthesis, arachidonic acid metabolism, and eicosanoid synthesis pathways were among the most enriched due to HPLF versus CON in MFP and were inhibited. These responses suggest that, in PAR, doubling nutrient intake from standard feeding rates inhibited energy metabolism and activity of oxidative pathways that partly serve to protect cells against oxidative stress. The MFP in those heifers appeared to decrease production of lipid-derived metabolites that may play roles in signaling pathways within the adipocyte. Overall, results indicated that prepubertal/preweaned mammary transcriptome is responsive to long-term enhanced nutrient supply to achieve greater growth rates before weaning. The biological significance of these results to future milk production remains to be elucidated.

Effect of milk fraction on concentrations of cephapirin and desacetylcephapirin in bovine milk after intramammary infusion of cephapirin sodium.

Clinical mastitis in dairy cows is commonly treated with intramammary (IMM) antimicrobial agents. Pharmacokinetic data are used to design treatment regimens and determine withholding times. In some pharmacokinetic studies, investigators measure antimicrobial concentrations in foremilk, whereas in others, they use bucket milk or do not specify the milk fraction sampled. Our objective was to compare antimicrobial concentrations in foremilk, bucket milk, and strippings after IMM treatment of six healthy Holsteins. One mammary gland/cow was infused with 200 mg of cephapirin (CEPH) after each of the two milkings, using different milking frequencies and treatment intervals in a randomized crossover design. Treated glands were sampled at the first milking following each infusion. Antimicrobial concentrations in milk were measured using HPLC/MS/MS. CEPH concentration was higher in foremilk (geometric mean 44.2 microg/mL) than in bucket milk (15.7 microg/mL) or strippings (18.5 microg/mL), as it was true for desacetylcephapirin (DAC) (59.5, 23.0, and 30.2 microg/mL, respectively). This finding, which was based on milk samples collected at the first milking after IMM infusion, suggests that pharmacokinetic data based on drug concentrations in foremilk may be misleading. Strippings were more representative of bucket milk than foremilk. The relationship between milk fraction and antimicrobial concentration should be investigated for other IMM antimicrobial agents. Meanwhile, it is essential that pharmacokinetic and residue studies report the fraction of milk that was analyzed.

Ideal amino acid balance for sows during gestation and lactation.

Improving efficiency of protein utilization is important for pregnant sows under restricted feed allowance and for lactating sows with limited feed intake. Sows have limited ability to support the growth of fetuses and mammary glands during late gestation and to support mammary growth and milk production, especially during first lactation period. A series of studies was conducted to characterize requirements and ideal ratios of AA for 1) fetal growth, 2) mammary gland growth of gestating sows, 3) maternal tissue gain of gestating sows, 4) mammary gland growth of lactating sows, and 5) maternal tissue gain of lactating sows. A total of 97 pregnant sows and their fetuses and a total of 174 lactating sows and their nursing piglets were used for these studies to collect fetal tissues, mammary tissues, and maternal tissues for AA analysis. Requirements and ideal ratios of AA for sows changed dynamically depending on stages of pregnancy. Suggested daily requirements for true ileal digestible Lys were 5.57 and 8.78 g, and relative ideal ratios for Lys:Thr:Val:Leu (on basis of AA weight) were 100:79:65:88 and 100:71:66:95 for d 0 to 60 and d 60 to 114 of gestation, respectively. Requirements and ideal ratios of AA for lactating sows changed dynamically depending on potential amounts of protein mobilization from maternal tissues, which are related to voluntary feed intake and milk production. Suggested ideal ratios for Lys:Thr:Val:Leu were 100:59:77:115 and 100:69:78:123 if BW losses of sows during 21 d of lactation are 0 and 33 to 45 kg, respectively. To optimize efficiency of dietary protein utilization by sows, the dietary AA content and ratios can be adjusted by stages of pregnancy (i.e., phase feeding) and by expected feed intakes or parities of sows during lactation (i.e., parity-split feeding) considering the dynamic changes in the requirements and ideal ratios of AA.

Internal controls for quantitative polymerase chain reaction of swine mammary glands during pregnancy and lactation.

High-throughput microarray analysis is an efficient means of obtaining a genome-wide view of transcript profiles across physiological states. However, quantitative PCR (qPCR) remains the chosen method for high-precision mRNA abundance analysis. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG), which is now, more than ever before, a fundamental step for accurate gene expression profiling. We mined mammary tissue microarray data on >13,000 genes at -34, -14, 0, 7, 14, 21, and 28 d relative to parturition in 27 crossbred primiparous gilts to identify suitable ICG. Initial analysis revealed TBK1, PCSK2, PTBP1, API5, VAPB, QTRT1, TRIM41, TMEM24, PPP2R5B, and AP1S1 as the most stable genes (sample/reference = 1 +/- 0.2). We also included 9 genes previously identified as ICG in bovine mammary tissue. Gene network analysis of the 19 genes identified AP1S1, API5, MTG1, VAPB, TRIM41, MRPL39, and RPS15A as having no known co-regulation. In addition, UXT and ACTB were added to this list, and mRNA abundance of these 9 genes was measured by qPCR. Expression of all 9 of these genes was decreased markedly during lactation. In a previous study with bovine mammary tissue, mRNA of stably expressed genes decreased during lactation due to a dilution effect brought about by large increases in expression of highly abundant genes. To verify this effect, highly abundant mammary genes such as CSN1S2, SCD, FABP3, and LTF were evaluated by qPCR. The tested ICG had a negative correlation with these genes, demonstrating a dilution effect in the porcine mammary tissue. Gene stability analysis identified API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Overall, results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG. Further, we showed that use of the same ICG from one organism might not be suitable for qPCR normalization in other species.

Effects of suckling intensity on milk yield and piglet growth from lactation-enhanced gilts.

The effects of suckling intensity on milk yield and piglet growth were determined when lactation capacity of the sow was enhanced through overexpression of a mammary-specific transgene, bovine alpha-lactalbumin. Lactational response to increased suckling stimulation was determined by fostering litters of the same age (d 1) or 7 d older (d 7) than the day of lactation to sows nontransgenic (control) or transgenic (TG) for bovine alpha-lactalbumin. Twenty first-parity gilts were allocated to 4 treatments dependent on gilt genotype and age of litter fostered (control d 1, control d 7, TG d 1, and TG d 7). Litters were standardized to 10 piglets within 24 h postpartum, and nonbirth piglets were fostered to gilts with an equal litter BW within age groups at 36 h postpartum. Milk yield was determined by the weigh-suckle-weigh method on d 6, 9, 12, 15, and 18 of lactation. Mean daily milk yield was greater (P = 0.031) for TG gilts compared with control gilts and tended to be greater (P = 0.056) for all gilts with d-7 piglets compared with those with d-1 piglets. Daily milk yield of TG d 7 gilts increased rapidly to peak at d 9 and was greater than milk yield of all control gilts at d 9 (P < 0.01), 12 (P < 0.02), and 15 (P < 0.02). Mean daily milk yield of TG d 7 gilts was 2.1 kg greater (P = 0.002) than for control d 7 gilts and 2.0 kg greater (P = 0.004) than for TG d 1 gilts. Daily milk yield of control d 1 gilts was not different from that of TG d 1 gilts (P = 0.49) or control d 7 gilts (P = 0.63). Piglet BW gain between d 3 and 6 was greater (P < 0.01) in the TG d 7 group than for all other groups and was greater (P < 0.05) than the control groups between d 6 and 9. No difference was found when comparing accumulated BW gain of the piglets between the day of age at foster (d 1 vs. 7; P = 0.606) or between the control d 1 and control d 7 groups (P = 0.759). Accumulated BW gain of piglets suckling TG d 7 gilts from d 3 through 9 was greater (P < 0.02) than that of the other groups and continued to be greater (P < 0.05) than that of either of the control groups through d 15. However, by d 15, accumulated BW gain of piglets suckling TG d 1 gilts was no longer different (P = 0.40) from that of the TG d 7 group and was greater (P < 0.05) than that of the control d 1 group. The enhanced lactation potential of these TG gilts synergized with suckling intensity to stimulate increased milk production during early lactation, resulting in increased piglet growth.

Role of suckling in regulating cell turnover and onset and maintenance of lactation in individual mammary glands of sows.

This study addressed the mechanisms by which suckling regulates cell turnover and onset and maintenance of lactation of individual mammary glands of sows. The effects of no, transient (through 12 to 14 h postpartum), or regular suckling of individual glands during d 0 to 6 of lactation were studied in 5 sows. Nonsuckling was obtained by taping the glands to prevent access to the nipples. Visual scores confirmed that regularly suckled glands maintained lactation, whereas transiently suckled and nonsuckled glands regressed during lactation. Mammary gland biopsies were collected on d -5, 1, 2, 4, and 6 relative to farrowing in order to evaluate the cell turnover and to quantify the transcription of genes potentially involved in mammary cell turnover and function. The proportion of proliferating cells was greatest prepartum (13.1%). After farrowing, the proportion of proliferating cells declined in all glands, then remained low (5.6%) in nonsuckled glands and increased (P < 0.01) from an average of 7.5% on d 1 to 9.9% on d 6 in regularly suckled and transiently suckled glands. Transcriptional data were analyzed using a gamma-distributed, generalized linear mixed model. Abundance of alpha-lactalbumin mRNA (P < 0.01) increased in regularly suckled glands within the first day of lactation and remained elevated, whereas the expression in taped glands remained at the prepartum level. Prolactin receptor mRNA abundance decreased (P < 0.001), and IGFBP-5 mRNA abundance increased (P < 0.01) in nonsuckled and transiently suckled glands after parturition compared with regularly suckled glands. Mammary mRNA abundances of IGF-I, IGF-II, type I IGF receptor, and caspase 3 were minimally or inconclusively affected by the suckling regimens. In conclusion, suckling during the first 12 to 14 h postpartum is insufficient to initiate and maintain lactation until 24 to 36 h postpartum but sufficient to induce mammary cell proliferation for at least 6 d postpartum. Furthermore, a high prolactin receptor transcription and a low IGFBP-5 transcription seem important for maintaining a functional mammary gland during lactation.

Estrogenic effects of genistein on reproductive tissues of ovariectomized gilts.

The soybean phytoestrogen genistein has a range of estrogenic actions demonstrated in various species; however, only limited research has been done to investigate its effects in swine. The objective of this study was to characterize the effects of a graded dose of genistein on estrogen-sensitive uterine and cervical tissues in ovariectomized gilts. Thirty-four postpubertal gilts were ovariectomized and assigned randomly to 1 of 6 treatment groups 15 d postovariectomy. Treatment groups received vehicle, estradiol benzoate (2 mg/d), or genistein (50, 100, 200, or 400 mg/d) via intramuscular injection at 12-h intervals for 10 d. Following the treatment period, gilts were euthanized, and uterine and cervical tissues were collected and processed for chemical or histological analysis. Uterine and cervical tissue mass, as indicated by wet, dry, and protein weights and total DNA content (expressed per 100 kg of BW), increased as the dosage of genistein increased (P < 0.001 for each regression). Uterine and cervical wet weights were increased by a dosage of 200 mg of genistein/d (P < 0.001 and P < 0.01, respectively) but not by 100 mg of genistein/d (P = 0.38 and P = 0.14, respectively) compared with those of control gilts. Height of epithelial cells lining the uterine glands and the lumen of uterus and cervix increased when gilts were treated with estradiol benzoate or 400 mg of genistein/d (P < 0.01). When the gilts were treated with estradiol benzoate or 400 mg of genistein/d, immunohistochemical staining demonstrated an increase in the percentage of cells that stained positive for progesterone receptor in the uterine glands and in the cells lining the vaginal cervix (P < 0.05). In gilts treated with 400 mg of genistein/d, the percentage of cells stained positive for proliferating cell nuclear antigen increased in the epithelium of the uterine glands, uterine lumen, and vaginal cervix (P < 0.05). Tissue growth was stimulated by genistein in a dosage-dependent manner, although no dosage of genistein induced a response as great as that of estradiol benzoate. Estrogen-sensitive tissues of the ovariectomized gilt, such as the cervix and uterus, are affected by injection of large dosages of the phytoestrogen genistein. The sensitivity of the uterus of the gilt to estrogenic substances makes it a potential model to examine the impact of environmental endocrine modulators on reproductive tissues.

Characterization of mammary gland development in pregnant gilts.

The purpose of this study was to quantify mammary gland (MG) growth during pregnancy in gilts and to determine the effect of anatomical location on gland growth. Size, composition, and histomorphology of MG were determined during gestation in 29 primigravid gilts. Gilts were allotted randomly to 6 slaughter groups: d 45 (n = 6), 60 (n = 4), 75 (n = 5), 90 (n = 4), 102 (n = 5), and 112 (n = 5) of gestation. Mammary glands were obtained at slaughter, and skin and extraneous fat pad were removed to obtain parenchymal MG tissue. Mammary glands were further separated into individual MG, and their locations were recorded. Individual MG were weighed and bisected in an approximate midsagittal section to measure cross-sectional area. Mammary glands were ground individually and pooled according to anatomical region: the first and second pairs of MG = anterior MG; the third, fourth, and fifth pairs of MG = middle MG; the sixth, seventh, and eighth pairs of MG = posterior MG. Contents of DM, CP, ether extract, and crude ash were measured. Wet weight, DM, CP, and ash content of total and individual MG increased (P < 0.01) between d 45 and 112 of gestation. Cross-sectional area of individual MG increased (P < 0.01) as gestation progressed. Percentage of CP and ash increased (P < 0.01), whereas percentage of ether extract decreased (P < 0.01) as gestation progressed. This inverse relationship between percentages of CP and ether extract (r = -0.999; P < 0.0001) was consistent with the histological shift from primarily an adipose tissue in early gestation to one containing extensive lobuloalveolar tissue in late gestation. Wet weight of middle MG was greater (P < 0.05) than that of posterior MG at d 102 and 112 of gestation, and amount of CP in middle MG was greater (P < 0.05) than that in anterior and posterior MG at d 102 and 112 of gestation, indicating that middle MG grow faster than other MG during late gestation. Rates of wet weight gain and protein accretion were accelerated (P < 0.01) after d 74 and 75 of gestation, respectively, indicating the importance of MG growth during the last trimester of gestation. The increase in rate of protein accretion after d 75 indicates a greater protein requirement for MG growth during later gestation.

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.

Quantification of mammary gland tissue size and composition changes after weaning in sows.

The objectives of this study were to characterize the tissue compositional changes in porcine mammary glands after weaning and to determine whether administration of estradiol alters the profile of these tissue changes. Forty-five primiparous sows were assigned randomly to one of two treatment groups after weaning, control or estrogen treated. Estrogen-treated sows received twice-daily injections of estradiol-17beta (0.125 mg/kg of BW); control sows received vehicle injections. Sows were weaned at d 21 of lactation and killed on either d 0 (d of weaning; n = 5) or on d 2, 3, 4, 5, or 7 after weaning (n = 4 per treatment on each day). Teat order relative to suckling behavior was observed on the day before weaning to determine which mammary glands the piglets suckled. Suckled and non-suckled glands were identified from the teat order observation, and individual mammary glands were collected at slaughter. Mammary glands were trimmed of skin and extraneous fat pad, individually weighed, and bisected to measure cross-sectional area. The remaining half of each gland was ground and stored at -20 degrees C for chemical analyses. Frozen tissue was used for measuring tissue DNA, DM, protein, fat, and ash contents. Suckled mammary glands of sows undergo significant and dramatic changes during the initial 7 d after weaning, with significant changes occurring even by d 2 after weaning. Mean cross-sectional area of parenchymal tissue in suckled mammary glands decreased from 59.7 +/- 2.1 cm2 on the day of weaning to 26.8 +/- 2.3 cm2 by d 7 after weaning (P < 0.0001). Mammary gland wet weight decreased from 485.9 +/- 22.0 g on the day of weaning to 151.5 +/- 24.8 g by d 7 after weaning (P < 0.0001), whereas DNA decreased from 838.8 +/- 46.2 g on the day of weaning to 278.4 +/- 52.5 g by d 7 after weaning (P < 0.0001). The changes in gland wet weight and DNA during the period of mammary gland involution in the sow represent loses of over two-thirds of the parenchymal mass and nearly two-thirds of the cells that were present on the day of weaning. Estrogen treatment did not affect overall mammary involution during the first 7 d after weaning. Mammary glands that were not suckled during lactation had no further loss of parenchymal tissue during the first 7 d after weaning. Mammary gland involution in the sow is a rapid process and is probably irreversible within 2 or 3 d after weaning.

Sequence and expression of the FcRn in the porcine mammary gland.

Transport of immunoglobulin G across epithelial cell barriers is thought to occur by a system involving the Fcgamma receptor called the neonatal Fc receptor (FcRn). The FcRn may also play a role in IgG transport in the mammary gland. To determine the presence of FcRn in the porcine mammary gland, biopsies were taken from glands 3 days prepartum and on the day of farrowing. The full length porcine FcRn cDNA sequence was obtained by rapid amplification of cDNA ends and determined to be 1557 base pairs in length that codes for a 359 amino acid peptide. Expression of FcRn mRNA in the porcine mammary gland was determined by reverse transcriptase-PCR and revealed that the mRNA is present prepartum and on the day of farrowing. These results indicate that the FcRn is expressed in porcine mammary tissue and are consistent with the hypothesis that FcRn may have a role in mammary gland immunoglobulin transport during colostrogenesis.

Lactational performance of first-parity transgenic gilts expressing bovine alpha-lactalbumin in their milk.

The goal of this study was to determine whether the presence of the bovine alpha-lactalbumin transgene in first-lactation gilts enhances lactational performance and litter growth. Transgenic and sibling nontransgenic gilts were bred to nontransgenic boars. Litters were standardized to 10 piglets within 24 h of farrowing. Milk production was measured by the weigh-suckle-weigh method on d 3, 6, 9, and 12 of lactation. Bovine alpha-lactalbumin was present in the colostrum and milk of transgenic gilts throughout lactation. The expression of the transgene was associated with alterations in composition of mammary secretions, especially in early lactation. Lactose concentrations were greater (P < 0.05) in mammary secretions of transgenic gilts during the first 12 h postpartum compared with controls. In contrast, total solids concentration in mammary secretions from transgenic gilts were lower (P < 0.05) relative to controls during the first 6 h postpartum. Transgenic gilts produced more milk than controls on d 3, 6, and 9 of lactation (P < 0.01). By d 12, differences in milk production between transgenic and control sows were no longer different. Lactose intake by transgenic-reared litters was greater than lactose intake by control-reared litters on d 6 of lactation (P < 0.05). Total solids intake was significantly greater (P < 0.05) by transgenic-reared litters on d 3 and 6 compared to control-reared litters. The day x genotype interaction on litter weight gain after birth was highly significant (P = 0.011), with transgenic-reared litters gaining weight at a greater rate than control-reared piglets. Expression of the transgene was associated with increased milk production in lactating gilts and increased growth of transgenic-reared piglets. Increased lactose synthesis in response to the presence of the transgene may result in increased milk production in early lactation, leading to increased milk component intake by transgenic litters, and ultimately to increased growth of litters reared by first-parity transgenic gilts.

The regression of unsuckled mammary glands during lactation in sows: the influence of lactation stage, dietary nutrients, and litter size.

During lactation in the sow, mammary glands that are not regularly suckled undergo regression. This study characterizes the regression of unsuckled mammary glands and how that regression is affected by dietary nutrients and litter size. Sixty-nine primiparous sows were fed one of four diets containing combinations of two protein levels (32 or 65 g lysine/d) and two energy levels (12 or 17.5 Mcal ME/d) during lactation. Litter size was adjusted to 10. Sows were killed on d 0, 5, 10, 14, 21, or 28 of lactation. In another experiment, twenty-eight primiparous sows were allotted to have different litter sizes and were killed on d 21 of lactation. The day before slaughter, teat order of each litter was observed. After death, mammary glands were removed and dissected. Skin and extraneous fat pads were removed from the mammary glands and individual glands were separated. Each gland was weighed, cut in half to measure cross-sectional area, and ground for chemical analysis. The amounts of dry tissue, protein, fat, ash, and DNA were measured. Only glands observed to be unsuckled were included in the results. Regression of unsuckled mammary glands occurred rapidly during the first 7 to 10 d of lactation, as indicated by a decline in wet weight, dry weight, protein, fat, DNA, and cross-sectional area. The rate of regression was slowed after the early lactation period. The rate of regression of unsuckled glands was affected by dietary nutrient levels. Dietary energy level affected (P < 0.05) the decline in wet and dry weights, protein, fat and DNA content, and cross-sectional area, whereas dietary protein level affected (P < 0.05) the decline in dry weight and fat content. At d 5 of lactation, the wet weight of unsuckled mammary glands in sows fed the high-energy high-protein diet was 91% greater (P < 0.05) than in sows fed the low-energy low-protein diet. Effects of litter size on size and composition of unsuckled glands were not significant by d 21 of lactation. Unsuckled mammary glands regress rapidly during early lactation, and the rate of regression is affected by dietary nutrient intake.

Cellular uptake of taurine by lactating porcine mammary tissue.

Milk taurine plays a critical role in neonatal development. Taurine uptake in lactating sow mammary tissue has not been characterized previously. The kinetic properties, ion dependence and substrate specificity of taurine uptake were characterized in mammary tissue collected from lactating sows at slaughter. Tissue explants were incubated in an isosmotic physiologic buffer with [3H]taurine tracer to measure taurine uptake. Taurine uptake was dependent upon the presence of extracellular sodium and chloride ions, which is consistent with the co-transport of sodium and chloride with taurine. Uptake was not dependent upon ion exchange mechanisms or upon furosemide-sensitive ion co-transport. Taurine uptake was saturable and exhibited an apparent Km of 20 microM and a V(max) of 386 micromol/kg cell water/30 min. Substrate specificity studies indicated a strong interaction of beta-amino acids with the taurine transport system. Taurine transport in lactating sow mammary tissue is therefore a high affinity, sodium-dependent mechanism specific for beta-amino acids, and is analogous to sodium-dependent taurine uptake in other tissues. The high affinity and high specificity of the taurine uptake system allows for concentration of taurine within the mammary cell and is ultimately responsible for provision of taurine required for neonatal development.

Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk.

Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats. Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S. aureus mastitis. In the study reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S. aureus (isolates from bovine mastitis). A specific amplicon of 270 bp was generated as predicted. Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S. aureus detection in milk from experimentally infected cows from 65 to 80%. Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples from cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed. It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as but faster than conventional milk bacteriological culture techniques and is highly specific. The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S. aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.

Cellular uptake of valine by lactating porcine mammary tissue.

The cellular uptake of branched-chain amino acids in mammary tissue is important for understanding their role in milk synthesis in the sow. This study characterized the kinetic properties and substrate specificity of the valine uptake system in the porcine mammary gland. Mammary tissue was collected from lactating sows at slaughter and tissue explants were incubated in media containing isosmotic salt and amino acids of interest, plus [3H]valine tracer. Valine uptake was time-dependent and was dependent on the presence of sodium, as indicated by a reduction in uptake when sodium in the medium was replaced by choline. The valine transport system in porcine mammary tissue had a Km of 0.64 mM, a Vmax of 1.84 mmol-kg cell water(-1) 30 min(-l), and a Kd (diffusion constant) of 1.16 L x kg cell water(-1) x 30 min(-1). Valine uptake was inhibited by leucine and alpha-aminoisobutyric acid and by high concentrations of L-alanine, L-lysine, cycloleucine, L-glutamine, and L-methionine, but not by 2-(methyl-amino)-isobutyric acid. This transport system is the primary system responsible for uptake of valine, and probably other branched-chain amino acids, in lactating sow mammary tissue. Physiological concentrations of valine in the blood are below the Km of the specific valine transport system and well below the diffusion uptake capabilities. The kinetic parameters of this valine transport system should not be limiting to valine uptake for milk protein synthesis. However, competition of valine uptake with branched-chain amino acids, as well as with other amino acids, may affect valine uptake in lactating tissue.

Growth of nursing pigs related to the characteristics of nursed mammary glands.

The purpose of this study was to determine growth performance of nursing pigs in relationship to teat order and to observe teat preference by pigs. In the first experiment, litter size of 13 primiparous sows was adjusted to 9 (8.7 +/- 1.5) pigs and teat order of each litter was observed on the day before slaughter. Another group of eight sows was killed on d 0 (within 12 h after farrowing). In the second experiment, litter size was adjusted to 9 (8.9 +/- 1.4) pigs for 20 primiparous sows and teat order for each litter was observed 1 d before slaughter. The weights of sows and individual pigs were recorded at farrowing, weekly, and on the day before slaughter. Mammary glands were collected at slaughter on d 21 of lactation and trimmed of skin and the extraneous fat pad. Individual glands were separated, weighed, and ground for measurement of dry matter, dry fat-free tissue, protein, fat, ash, and DNA contents. Middle mammary glands had the greatest wet weight among glands obtained within 12 h after weaning (P < .05). For sows completing the 21 d lactation, only glands known to have been nursed were included in the data sets. Greater than 60% of the first four pairs of mammary glands were nursed, and less than 40% of the seventh and eighth glands were nursed by pigs during lactation. Pigs that nursed the first five pairs of anterior glands gained faster than pigs nursing the remaining glands. The first five pairs of anterior glands had greater wet and dry weights, and greater protein and DNA contents compared with the remaining glands. Pigs that nursed heavier glands gained weight faster (r = .68, P = .0001), and those heavier glands contained greater amounts of protein (r = .98, P = .0001) and DNA (r = .66, P = .0001). Variation in weight gain of pigs nursing the anterior and middle glands was not statistically significant. The functional superiority of anterior and middle glands was positively correlated with body weight gain of nursing pigs.

Lysine uptake by mammary gland tissue from lactating sows.

Kinetic properties and substrate specificity of the lysine transport system in porcine mammary gland were studied using mammary tissue explants from nine lactating sows. Sodium dependence of lysine uptake was determined by replacing sodium in the medium with choline. Kinetic parameters of lysine uptake were determined using lysine concentrations from 5 microM to 5.12 mM. Competition of lysine uptake by other amino acids was determined using the cationic amino acids, arginine and ornithine, and using other essential amino acids. Transport of lysine was time-dependent and was unaffected by replacing sodium with choline. Lysine uptake occurred by a transport mechanism with a Km of approximately 1.4 mM and a Vmax of 7.9 mmol x kg cell water(-1) x 30 min(-1). Lysine uptake was inhibited by arginine and ornithine and by high concentrations of L-alanine, L-methionine, L-leucine, cycloleucine, and D-lysine, but not by 2-(methylamino)-isobutyric acid. This transport mechanism is the primary system responsible for uptake of cationic amino acids in lactating sow mammary tissue. The relatively high Km, compared with physiological blood concentrations of lysine, indicates that the kinetic properties of the lysine transport system should not be limiting to milk protein synthesis. Transmembrane transport of lysine by lactating sow mammary tissue should be a direct function of plasma concentrations. However, interactions of other amino acids with the uptake system may affect lysine uptake.