Fatty acid profile of phospholipids and sphingomyelin in milk and regulation of sphingomyelin synthesis of mammary glands in cows receiving increasing levels of crushed sunflower seeds.

The objective of this study was to investigate the effect of increasing dietary supplementation of crushed sunflower seed (CSS) in the diet of dairy cows on the fatty acid (FA) composition of phospholipids and sphingomyelin in milk, and on mammary transcription of genes that are important for sphingomyelin de novo synthesis. Four groups of 6 cows received diets supplemented with CSS at 0% (control), or 5, 10, or 15% of dry matter for a 5-wk experimental period. Milk samples and mammary biopsies were collected at the end of the experiment. Phospholipid concentration in milk fat decreased linearly with CSS supplementation. Sphingomyelin concentration in milk fat was unaffected by CSS supplementation. Daily yield of phospholipids decreased linearly with CSS supplementation. Daily yield of sphingomyelin was not significantly affected. The CSS supplementation linearly increased the proportion of monounsaturated FA in milk phospholipids. The major isomer incorporated into phospholipids was C18:1 (n-9 cis), which showed a linear increase with CSS supplementation. The C22:0 proportion in sphingomyelin increased linearly with CSS supplementation and constituted between 15.2 to 25.4% of total FA in sphingomyelin. However, CSS supplementation linearly decreased C23:0 sphingomyelin. Mammary transcription of serine palmitoyl transferase, long chain subunit 1 and subunit 2, the rate-limiting enzymes in ceramide synthesis, showed a linear decrease with increasing CSS supplementation. In conclusion, the data showed that dietary supplementation of CSS linearly increased the proportion of unsaturated FA and monounsaturated FA in milk phospholipids with no effect on phospholipid concentration. In addition, CSS supplementation linearly decreased n-3 polyunsaturated fatty acid proportion in sphingomyelin. The results further showed that mammary transcription of important genes for sphingomyelin de novo synthesis is regulated by lipid supplementation.

Late gestation undernutrition can predispose for visceral adiposity by altering fat distribution patterns and increasing the preference for a high-fat diet in early postnatal life.

We have developed a sheep model to facilitate studies of the fetal programming effects of mismatched perinatal and postnatal nutrition. During the last trimester of gestation, twenty-one twin-bearing ewes were fed a normal diet fulfilling norms for energy and protein (NORM) or 50% of a normal diet (LOW). From day 3 postpartum to 6 months (around puberty) of age, one twin lamb was fed a conventional (CONV) diet and the other a high-carbohydrate-high-fat (HCHF) diet, resulting in four groups of offspring: NORM-CONV; NORMHCHF; LOW-CONV; LOW-HCHF. At 6 months of age, half of the lambs (all males and three females) were slaughtered for further examination and the other half (females only) were transferred to a moderate sheep diet until slaughtered at 24 months of age (adulthood). Maternal undernutrition during late gestation reduced the birth weight of LOW offspring (P<0·05), and its long-term effects were increased adrenal size in male lambs and adult females (P<0·05), increased neonatal appetite for fat-(P=0·004) rather than carbohydrate-rich feeds (P<0·001) and reduced deposition of subcutaneous fat in both sexes (P<0·05). Furthermore, LOW-HCHF female lambs had markedly higher visceral:subcutaneous fat ratios compared with the other groups (P<0·001). Postnatal overfeeding (HCHF) resulted in obesity (.30% fat in soft tissue) and widespread ectopic lipid deposition. In conclusion, our sheep model revealed strong pre- and postnatal impacts on growth, food preferences and fat deposition patterns. The present findings support a role for subcutaneous adipose tissue in the development of visceral adiposity, which in humans is known to precede the development of the metabolic syndrome in human adults.

Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells--implications for milk cholesterol secretion.

The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play an important role in cellular cholesterol homeostasis, but their function in mammary gland (MG) tissue remains elusive. A bovine MG model that allows repeated MG sampling in identical animals at different functional stages was used to test whether 1) ABCA1 and ABCG1 protein expression and subcellular localization in mammary epithelial cells (MEC) change during the pregnancy-lactation cycle, and 2) these 2 proteins were present in milk fat globules (MFG). Expression and localization in MEC were investigated in bovine MG tissues at the end of lactation, during the dry period (DP), and early lactation using immunohistochemical and immunofluorescence approaches. The presence of ABCA1 and ABCG1 in MFG isolated from fresh milk was determined by immunofluorescence. The ABCA1 protein expression in MEC, expressed as arbitrary units, was higher during the end of lactation (12.2±0.24) and the DP (12.5±0.22) as compared with during early lactation (10.2±0.65). In contrast, no significant change in ABCG1 expression existed between the stages. Throughout the cycle, ABCA1 and ABCG1 were detected in the apical (41.9±24.8 and 49.0±4.96% of cows, respectively), basal (56.2±28.1 and 54.6±7.78% of cows, respectively), or entire cytoplasm (56.8±13.4 and 61.6±14.4% of cows, respectively) of MEC, or showed combined localization. Unlike ABCG1, ABCA1 was absent at the apical aspect of MEC during early lactation. Immunolabeling experiments revealed the presence of ABCA1 and ABCG1 in MFG membranes. Findings suggest a differential, functional stage-dependent role of ABCA1 and ABCG1 in cholesterol homeostasis of the MG epithelium. The presence of ABCA1 and ABCG1 in MFG membranes suggests that these proteins are involved in cholesterol exchange between MEC and alveolar milk.

Mammary remodeling in primiparous and multiparous dairy goats during lactation.

Milk production is generally lower but lactation persistency higher in primiparous (PP) than in multiparous (MP) goats. This may be related to differences in development and maintenance of mammary gland function, but the underlying mechanisms are not well understood. The present study aimed to elucidate whether differences in lactational performance between PP and MP mammary glands are related to the time course of development and maintenance, not only of the mammary epithelial cell (MEC) population, but also of the mammary vasculature that sustains synthetic activity. Mammary biopsies were obtained from both mammary glands of 3 PP and 6 MP (>or=2 parity) dairy goats at parturition (d 1), d 10, 60, and 180 of lactation. Gene transcription relating to MEC turnover and vascular function was quantified by real-time reverse transcription-PCR, mammary morphology was characterized (quantitative histology), and cell turnover was determined (terminal deoxynucleotidyl transferase dUTP nick end labeling assay and Ki-67). Primiparous glands showed higher expression for the genes involved in angiogenesis; namely, vascular endothelial growth factor receptor 2, and angiopoietin 1 and 2 and their receptor, a few days after parturition (d 10). Primiparous glands also had higher rates of MEC proliferation in early lactation. It therefore appears that initiation of lactation is associated with development and growth of the mammary gland into early lactation, which continues for a longer period in PP compared with MP glands. In addition, MEC survival was found to be higher in PP glands throughout lactation, and MEC in PP glands underwent more extensive differentiation. This could explain the reported flatter lactation curve and higher lactation persistency in PP glands. Although some of the genes included in this study were differentially expressed in PP and MP glands during the course of lactation, it was not possible to identify any specific genomic factor(s) that could account for the differences between PP and MP glands with respect to mammary development and MEC survival during lactation. It remains to be established why parity number affects MEC and vascular development and survival during lactation, and, in particular, which regulatory mechanisms are involved.

Continuous lactation effects on mammary remodeling during late gestation and lactation in dairy goats.

The present study aimed to 1) elucidate whether continuous milking during late gestation in dairy goats negatively affects mammary remodeling and hence milk production in the subsequent lactation, and 2) identify the regulatory factors responsible for changes in cell turnover and angiogenesis in the continuously lactating mammary gland. Nine multiparous dairy goats were used. One udder half was dried off approximately 9 wk prepartum (normal lactation; NL), and the other udder half of the same goat was milked continuously (continuous lactation; CL) until parturition or until the half-udder milk yields had dropped to below 50 g/d. Mammary biopsies were obtained from each udder half just before the NL gland was dried off (before dry period), within the first 2 wk after drying-off (early dry period, samples available only for NL glands), in the mid dry period, within the last 2 wk before parturition (late dry period), and at d 1 (the day of parturition), 3, 10, 60, and 180 of lactation. Mammary morphology was characterized in biopsies by quantitative histology, and cell turnover was determined by immunohistochemistry (terminal deoxynucleotidyl transferase dUTP nick end labeling and Ki-67). Transcription of genes encoding factors involved in mammary epithelial cell (MEC) turnover and vascular function was quantified by quantitative reverse transcription PCR. Results demonstrated that omitting the dry period was possible in goats but was not as easy as claimed before. Renewal of MEC was suppressed in CL glands, which resulted in a smaller MEC population in the subsequent lactation. At the time of parturition (and throughout lactation), the mammary glands subjected to CL had smaller alveoli, more fully differentiated MEC, and a substantially larger capillary fraction compared with NL glands. The continuously lactating gland thus resembled a normally lactating gland in an advanced stage of lactation. None of the studied genomic factors could account for these treatment differences. The described characteristics in CL glands compared with NL glands indicated a gland maintained in lactation for a longer period.

Differential expression and localization of lipid transporters in the bovine mammary gland during the pregnancy-lactation cycle.

The transport of lipids across mammary gland epithelial cells (MEC) determines milk lipid content and composition. We investigated the expression of lipid transporters and their regulators in comparison to blood metabolites during lactation and dry period (DP) in dairy cows. Repeated mammary gland biopsies and blood samples were taken from 10 animals at 7 stages of the pregnancy-lactation cycle. Expression levels of the specific mRNAs were determined by quantitative reverse transcription-PCR, whereas ABCA1 was localized by immunohistochemistry. Blood serum metabolites were determined by common enzymatic chemistries. Elevated mRNA profiles of ABCA1 and ABCA7 were found during DP as compared with lactation and were inversely associated with blood cholesterol levels. Elevated levels of ABCG2, NPC1, SREBP1, SREBP2, LXR alpha, and PPAR gamma were found postpartum, whereas ABCG1 did not differ between the functional stages of the mammary gland. The ABCA1 protein was localized in MEC and showed differential activity between DP and lactation suggesting a role of ABCA1 in the removal of excess cellular cholesterol from MEC during the DP. The expression profiles of ABCA7 and NPC1 may reflect a role of these transporters in the clearance of apoptotic cells and the intracellular redistribution of cholesterol, respectively. Regulation of lipid transporters in the mammary gland is partially associated with transcription factors that control lipid homeostasis.

Effect of forage on the content of phyto-oestrogens in bovine milk.

Phyto-oestrogens are believed to have a range of beneficial effects on predominant Western diseases. A few studies on phyto-oestrogens in milk exist and show that the composition can be affected by feeding. Therefore, the aim was to study how feeding of lucerne and grass/clover silages (GCSs) affects the concentration of phyto-oestrogens in bovine milk. Sixteen Danish Holstein cows were assigned to a 4 × 4 latin square design with four cows per treatment per period of 3 weeks. The four treatment diets were lucerne silage (LS), 2/3 lucerne silage and 1/3 maize silage (2/3LS), 1/3 lucerne silage and 2/3 maize silage (1/3LS) and GCS. Milk was collected at the end of each period and feed samples on day 6, 13 and 20 in each experimental period. Milk and pooled feed samples were analysed for the concentration of isoflavones, coumestans and lignans. The content of isoflavones was higher and the content of coumestrol lower in the GCS diet than in LS, 2/3LS and 1/3LS diets. For the LS, 2/3LS and 1/3LS diets, the concentration of coumestrol and secoisolariciresinol increased with the proportion of lucerne while the concentration of isoflavones was similar across the diets. The concentrations of the formononetin, daidzein and equol in the milk were significantly higher for the GCS diet than for the LS, 2/3LS and 1/3LS diets. In particular, the concentration of equol was 62-291 times higher for GCS. The concentration of coumestrol was significantly lower for the GCS diet compared to the LS, 2/3LS and 1/3LS diets. No pattern for the concentration of lignans was observed. In conclusion, a high concentration of isoflavones, particularly equol, and a low concentration of coumestrol were measured in the milk from the GCS diet. In contrast, a low concentration of isoflavones and a high concentration of coumestrol were measured in the milk from the LS, 2/3LS and 1/3LS diets. However, the concentration of phyto-oestrogens in bovine milk is low compared to food sources rich in phyto-oestrogens but the high concentration of equol could possibly be of therapeutic importance.

Cellular mechanisms in regulating mammary cell turnover during lactation and dry period in dairy cows.

The mechanisms involved in regulating mammary cell turnover during the pregnancy-lactation cycle in dairy cows are unclear. The objective of present experiment was to describe expression of genes encoding proteins known to be involved in pathways regulating mammary cell proliferation, apoptosis, differentiation, cell survival, and tissue remodeling. Mammary gland biopsies were taken 7 times during the pregnancy-lactation cycle of 10 dairy cows, and samples were analyzed by immunohistochemistry and real-time PCR. Cell proliferation was greatest during the dry period and apoptosis was high in early dry period and early lactation. Based on Fas (tumor necrosis factor receptor superfamily member 6), Fas ligand, and caspase-3, caspase-8, and caspase-9 gene expression, no indication was found of a stage-dependent shift between the extrinsic and intrinsic pathways leading to apoptosis. Gene expression of microsomal glutathione S-transferase (mGST) did not vary significantly, whereas B-cell leukemia/lymphoma 2 (Bcl-2) and BCL2-associated X protein (Bax) gene expression was greatest during the dry period and early lactation and coincided with high cell turnover. Gene expression of early response genes c-Fos, c-Jun, and c-Myc correlated to neither rate of cell proliferation nor plasma concentration of insulin-like growth factor (IGF)-I and insulin. Gene expression of nuclear factor of kappa light chain gene enhancer in B-cells (NFkappaB) and NFkappaB inhibitor alpha was greatest in the periparturient period, and NFkappaB gene expression coincided with an anticipated need for cell survival factors. Expression of transforming growth factor beta (TGF-beta) receptor 1 and 2 mRNA was greatest in early lactation, whereas TGF-beta1 did not vary significant during the pregnancy-lactation cycle. Even though our results on the TGF-beta system did not comply with other studies, the gene expression pattern of the TGF-beta receptors indicates a role in regulating apoptosis in early lactation. Signal transducer and activator of transcription 5 (STAT5) gene expression was high in the periparturient period, which suggests a role for STAT5 in regulation of mammary cell proliferation and differentiation in dairy cows. Expression of tissue-plasminogen activator, plasminogen activator inhibitor-1, and IGF binding protein 5 genes was greatest in early lactation, suggesting a role for IGF binding protein 5 in coordinating regulation of apoptosis and tissue remodeling.

Effect of pregnancy and feeding level on cell turnover and expression of related genes in the mammary tissue of lactating dairy cows.

Milk yield is reduced by pregnancy, and the present experiment was conducted to study the biological basis for the negative effect of pregnancy on milk yield. A total of 16 dairy cows were fed at either a normal or a low feeding level (eight cows per treatment), and half of them were inseminated after approximately 3 months of lactation and the other half were not inseminated. Mammary biopsies were taken at approximately 9 months of lactation. The milk yield of pregnant cows was reduced by 2.6 kg/day, and lactation persistency was reduced already from the time of insemination. Low feeding level reduced the milk yield by 9.8 kg/day from week 8 to week 39 of lactation, whereas no interaction between pregnancy and feeding level was found. Cell proliferation (Ki-67) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL) were unaffected by feeding level, and pregnancy tended to reduce cell proliferation but had no effect on apoptosis. Reduced cell proliferation may explain the reduced lactation persistency in pregnant cows. Transcription of oestrogen receptor α, progesterone receptor A and B, and long and short isoforms of the prolactin receptor were higher in pregnant cows compared with non-pregnant cows. Feeding level did not mediate changes in transcription of genes. Transcription of other cell-turnover-related genes (IGF-I, IGF binding protein-5, caspase-3) as well as genes related to the secretory activity of the cells (α-lactalbumin and acetyl CoA carboxylase α) was not affected by pregnancy or by feeding level.

Cell turnover and activity in mammary tissue during lactation and the dry period in dairy cows.

Milk yield of the dairy cow follows a pattern termed the lactation curve. We have investigated the cellular background for this pattern. Seven mammary biopsies were obtained from each of 10 cows: at the end of lactation (d 347, equal to d 77 before next parturition); during the dry period at d 48 (4 d after dry off); 16 d before parturition; and during lactation at d 14, 42, 88, and 172. The fraction of proliferating (staining positive for Ki-67) alveolar cells was higher during the dry period (8.6%) than during lactation (0.5%). The fraction of apoptotic (staining positive by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) alveolar cells was higher immediately after dry off (0.37%) and in early lactation (0.76%) than during other periods (0.15%). The enzyme activities of fatty acid synthetase, acetyl CoA-carboxylase, and galactosyl transferase were approximately 12-, 11-, and 4-fold higher, respectively, during lactation than during the dry period. In conclusion, mammary cell proliferation is substantial in a period near parturition but otherwise low, and apoptosis is elevated at dry off and in early lactation. The increase in apoptosis in early lactation may be due to discarding nonfunctional or senescent cells or to removal of a surplus of newly synthesized cells. The activity of selected enzymes central for milk synthesis is probably not limiting for milk production.

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.

Leptin does not act directly on mammary epithelial cells in prepubertal dairy heifers.

The mammary gland of prepubertal dairy heifers consists of parenchyma expanding into the stroma, a matrix of connective and adipose tissue. High planes of nutrition increase stromal mass, but inhibit growth of parenchyma. The parenchyma consists of epithelial cells proliferating in response to growth factors such as insulin like growth factor-I (IGF-I). These observations have led to the hypothesis that elevated planes of nutrition increase leptin production, which in turn inhibits IGF-I-mediated epithelial cell proliferation. To assess this possibility, heifers were offered planes of nutrition sustaining average daily gains of 715 g/d (normal; NP) or 1,202 g/d (high; HP) from 42 d of age until slaughter at 240 kg. At slaughter, HP heifers had 2-fold greater plasma leptin concentration and 3-fold greater leptin mRNA abundance in mammary stroma and parenchyma. To assess the causal nature between leptin and parenchymal development, the induction of signaling events and functional responses in the MAC-T cell line and in primary mammary epithelial cells by leptin was examined. Leptin did not induce phosphorylation of signal transducers and activators of transcription (STAT)3, STAT5, extracellular signal-regulated kinase (ERK1/2), or AKT/Protein kinase B. Consistent with its inability to signal, leptin did not alter basal- or IGF-I-stimulated thymidine incorporation or increase suppressors of cytokine signaling 3 (SOCS3) expression in these cells. Transcripts corresponding to the short leptin receptor form were present in mammary tissue, but those corresponding to the long signaling form were not detected in either mammary tissue or cells. In conclusion, elevated planes of nutrition increase leptin synthesis in mammary stroma, but leptin does not act directly on bovine mammary epithelial cells.

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

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 gamma-distributed generalized linear mixed model. Mammary transcription of alpha-lactalbumin was high in suckled glands the day after farrowing, indicating onset of lactation, but was downregulated after 1 d of milk stasis. The prolactin receptor mRNA was downregulated and IGFBP-5 mRNA was upregulated within 1 d of milk stasis. The downregulation of alpha-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 alpha-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.

Mammary cell turnover and enzyme activity in dairy cows: effects of milking frequency and diet energy density.

The aim of the study was to investigate the effects of diet energy density and milking frequency on mammary cell turnover and synthetic capacity in dairy cows. Experiment 1 used 20 dairy cows. From d 4 to wk 16 postpartum, the cows were fed either a low-energy density or a high-energy density diet. From d 4 through wk 8, half of the cows in each group were milked 3 times (3x) or 2 times (2x) daily. From wk 9 to 16, all cows were milked 2 times daily. Mammary biopsies were obtained at wk 8 and 16 postpartum. In experiment 2, udders of 18 individual cows were milked diagonally 2x and 4x, and biopsies were obtained after 7 d. In experiment 1, cows on the low-energy density diet yielded 17 and 24% less milk during wk 3 to 8 and wk 11 to 16 postpartum, respectively. Furthermore, at 8 wk postpartum, mammary enzyme activities tended to be lower and mammary cell proliferation was lower in cows on the low-energy density diet. Three times daily milking during the first 8 wk postpartum resulted in 11% higher milk yield. Mammary cell turnover or enzyme activities were not significantly affected at 8 wk. The 3x milking for 8 wk resulted only in a transient carryover effect on milk yield and neither cell turnover nor enzyme activities were significantly affected at 16 wk postpartum. In experiment 2, mammary cell turnover and enzyme activity were unaffected after 7 d of 4x milking although milk yield increased by 18%. We conclude that nutrient restriction affects mammary cell turnover and possible enzyme activity, and that tuning of negative feedback loops in response to filling of the gland may be the dominating effects of changes in milking frequency.

Use of an immortalized bovine mammary epithelial cell line (MAC-T) to measure the mitogenic activity of extracts from heifer mammary tissue: effects of nutrition and ovariectomy.

The objectives of the experiment were (1) to determine whether MAC-T cells would accurately mimic the previously observed proliferative responses of primary mammary epithelial cells (MEC) to mammary tissue extracts from high and low-fed heifers and (2) to determine whether mammary tissue extracts from ovariectomized (OVX) heifers would have lower mitogenic activity than intact controls. Addition of mammary tissue extracts to cell culture media of MAC-T cells plated on plastic or collagen-coated plastic to a range of concentrations between 1 and 8% resulted in dose-dependent increases in cell proliferation. Furthermore, mammary tissue extracts from low-fed prepubertal heifers aged 9 months, stimulated significantly more proliferation of MAC-T cells, as measured by 3H-thymidine incorporation into DNA than mammary tissue extracts from high-fed heifers (40.6 cpm x 10(3) per well versus 21.9+/-1.8 cpm x 10(3) per well). These observations suggested that MAC-T cells would be a suitable alternative to primary MECs for measuring the mitogenic activity of mammary tissue extracts. Conversely, no difference was observed in the mitogenic activity of mammary tissue extracts from OVX or control heifers. Possibly, MAC-T cells provide a good model for nutrition- but not ovarian-induced changes in mammary growth. Alternatively, that reduction of in vivo mammary development following OVX did not result in reduced mitogenic activity of the mammary tissue extracts emphasizes that heifer mammary development is the result of complex interactions between local growth factors and systemic hormones.

Effects of growth hormone and feeding level on endocrine measurements, hormone receptors, muscle growth and performance of prepubertal heifers.

Prepubertal Friesian heifer calves (n = 24, initial BW = 195 +/- 5 kg) were assigned to a 2 x 2 factorial block design and used to evaluate the effects of daily GH treatment (0 or 15 mg/d) at either a low or a high feeding level in a 5-wk treatment period on endocrine measurements, hormone receptors, muscle growth, and overall performance. In the pretreatment period, a low feeding level was employed for all calves. During the treatment period, animals at the low feeding level had free access to a roughage-based mixture, whereas animals at the high feeding level had free access to a concentrate mixture and were offered 2 kg/d of the roughage-based mixture. Blood samples were collected weekly starting 3 wk before treatment. Longissimus (LM) and supraspinatus (SS) muscles were obtained at slaughter. Metabolizable energy intake was 81% higher, digestible CP intake was 140% higher, and ADG was 115% higher (all P < 0.001) at the high vs. low feeding level. Feed (DMI, ME, and protein) intake was not affected by GH treatment, but ADG was 18% higher (P < 0.13) in GH-treated than in control heifers at both feeding levels. Although of different magnitudes, the muscle anabolic effects of GH treatment and high vs. low feeding level were additive, and both treatments increased carcass weights (P < 0.02 and P < 0.001, respectively), LM (P < 0.05 and P < 0.001), and SS (P < 0.06 and P < 0.003). The anabolic effect of GH treatment was similar in both muscles, whereas the effect of feeding level was most pronounced in LM. Overall, GH treatment increased plasma GH, IGF-I (both P < 0.001), and IGFBP-3 (P < 0.02); however, GH treatment increased total IGF-I, free IGF-I, and IGFBP-3, and decreased IGFBP-2 mainly at the high feeding level (GH x feeding level interaction; P < 0.02, 0.01, 0.03, and 0.10, respectively). The high feeding level increased insulin, free and total IGF-I, and IGFBP-3 (all P < 0.001), but decreased GH and IGFBP-2 (both P < 0.001). High feeding increased type-1 IGF receptor density (P < 0.02), mainly in LM, in accordance with the largest anabolic response in this muscle, whereas GH treatment had no effect on type-1 IGF receptors. The results suggest that in skeletal muscle, the anabolic effects of exogenous GH are related to endocrine changes in the GH-IGF axis, whereas the effects of feeding level also seem to rely on IGF receptor density in the muscles.

Effect of feeding prepubertal heifers with a high oil diet on mammary development and milk production.

The objective of this study was to evaluate the effect of feeding prepubertal heifers a diet containing a high level of polyunsaturated fatty acids on mammary development and milk production. A total of 116 Holstein heifers were either fed a conventionally formulated concentrate or a high oil (HO) concentrate, using the same formulation but including 20% soybean oil, from birth to 6 mo of age. After 6 mo of age, all heifers were managed identically. Mammary gland development was evaluated on heifers slaughtered at 4 mo (n = 10) and 12 mo (n = 30) of age. Other heifers were bred when they reached 15 mo of age and milk production and feed intake were recorded every day from wk 4 to 18 of lactation. Feeding the high oil concentrate increased the concentration of linoleic acid in blood plasma (176%) and mammary fat pad (78%) at 4 mo of age and mammary fat pad (93%) at 12 mo of age. At 4 mo of age, mammary development was similar in both treatments. At 12 mo of age, total, parenchyma, and stroma weights of the mammary gland were not affected by treatments. However, lipid content was lower and concentration of DNA was higher in the parenchyma of heifers fed the high oil diet. Nevertheless, total parenchymal DNA and dry fat free tissue content did not reach statistical significance despite the fact that they were, respectively, 15 and 21% higher in HO heifers. Milk production and composition was not affected by treatments. In conclusion, feeding prepubertal heifers with a high oil concentrate slightly improved the mammary development but effects were too small to be translated into better lactating performances.

Milk production, nutrient utilization, and endocrine responses to increased postruminal lysine and methionine supply in dairy cows.

The effect of increased postruminal supply of lysine and methionine was investigated in a production trial involving 64 dairy cows in early lactation. Within each of two basal rations, based on either corn silage or grass silage, rations were either naturally deficient in lysine or fortified with 24 g of lysine in a rumen-protected form and naturally deficient in methionine or fortified with 12 g of methionine in a rumen-protected form. The data were analyzed separately for the four lysine and the four methionine treatment groups. Milk production, body weight gain, and plasma concentrations of insulin-like growth factor-I, bovine somatotropin, insulin, glucose, nonesterified fatty acids, and urea were monitored over a 12-wk period. Supplementation with protected methionine led to increases in milk fat and protein contents of 2.4 and 1.8 g/kg of milk, respectively. Supplementation with protected lysine or methionine numerically increased protein yield comparable to values reported in the literature, but the treatment effects were not statistically significant. Efficiency of utilization of absorbed amino acids for milk protein synthesis and efficiency of utilization of metabolizable energy for milk production were not significantly altered in response to increased postruminal lysine and methionine flow, but a numerically increased efficiency of utilization of total amino acids was observed. No significant effect of lysine or methionine supplementation was observed on endocrine parameters nor on plasma metabolite concentrations. However, across treatment groups, high milk yield was correlated with low plasma insulin-like growth factor-I concentrations (r = -0.44) and partially with low plasma nonesterified fatty acids concentration and insulin levels (r = -0.26), while body weight gain was negatively correlated (r = -0.33) with elevated plasma bovine somatotropin concentrations.

Differential effects of retinoids on proliferation of bovine mammary epithelial cells in collagen gel culture.

The effects of increasing concentrations of retinol, retinal and retinoic acid on proliferation of bovine mammary epithelial cells were investigated in collagen gel cultures. All retinoids significantly inhibited proliferation of mammary epithelial cells. The relative inhibitory potency of the retinoids was: retinoic acid > retinal > retinol. Maximal inhibition at 10 microg/ml corresponded to a 75-95% inhibition of proliferation obtained in basal medium. Retinol, retinal and retinoic acid also inhibited proliferation of cells growth-stimulated with insulin-like growth factor-I (IGF-I). Retinoids in highest concentrations (10 microg/ml) inhibited 68-85% of proliferation of cells obtained in culture medium containing 25 ng IGF-I/ml. Retinol and retinoic acid also inhibited proliferation of cells growth-stimulated by insulin and other growth factors from the IGF growth factor family (des(1-3)IGF-I and IGF-II), as well as growth factors from the epidermal growth factor family (EGF and TGF-alpha), with retinoic acid being more effective than retinol. At a concentration of 100 ng/ml retinol and retinoic acid inhibited respectively 24-38 and 44-52% of mammary cell proliferation stimulated by growth factors of the IGF family, and at 10,000 ng/ml, 61-71% of cell proliferation was inhibited. The growth-stimulating effect of insulin, EGF and TGF-alpha was inhibited 42-64% by retinol and retinoic acid at 100 ng/ml, and 64-84% at 10,000 ng/ml. The present results show that retinol, retinal and retinoic acid are potent inhibitors of bovine mammary epithelial cell proliferation. It is suggested that retinoids may have concentration-dependent roles in regulation of pubertal mammary growth and development, indicating that the milk yield potential of heifers may be affected by vitamin A status.

High body weight gain and reduced bovine mammary growth: physiological basis and implications for milk yield potential.

Available evidence concerning the relationship between growth rate, mammary growth and milk yield in heifers leads to these conclusions: 1) Increased growth rate due to high feeding level before puberty onset can lead to reduced pubertal mammary growth and reduced milk yield potential. 2) Increased growth rate due to high feeding level after puberty and during pregnancy have no effect on mammary growth and milk yield. 3) Higher body weight gain due to higher genetic potential for growth is positively related to milk yield. The negative effect of high feeding level before puberty occurs in all breeds, but the level of feeding causing reduced yield varies. Variation in responses between experiments suggests that feeding regimes that support high growth rates without negative effect on yield can be developed. A breakthrough most likely will originate from increased knowledge of the physiological relationship between nutrition and mammary development. Our investigations suggest that blood growth hormone (GH) is important for mammary development, and that the negative effect of high feeding level on mammary development may be due to reduced blood GH. GH, however, does not bind to mammary tissue. Experiments with exogenous GH suggest that GH acts on mammary tissue via IGF-I, but IGF-I is increased by high feeding level - not decreased as GH. This paradoxical relationship cannot be explained by changes in circulating IGF binding proteins. However, the sensitivity of mammary tissue to IGF-I is reduced by high feeding level, probably due to the action of locally produced binding proteins and/or growth factors.