Milk yield differences between 1× and 4× milking are associated with changes in mammary mitochondrial number and milk protein gene expression, but not mammary cell apoptosis or SOCS gene expression.

Milking frequency is known to affect milk production and lactation persistence in dairy cows. Despite this, the mechanisms underlying this effect are only partially understood. Previous work in dairy cows examining increases in milk yield due to increased milking frequency have identified changes in apoptosis and expression of genes regulating cytokine signaling. In addition, changes in mitochondrial biogenesis and function have been suggested to play a role during the lactation cycle in regulating milk production. The goal of this study was to test the hypothesis that, when maintained over an entire lactation, extreme differences in milking frequency would be reflected in differences in apoptosis, mammary mitochondrial number, and the mammary expression of genes known to inhibit cytokine signaling. Primiparous Holstein cows (n=6) were assigned to the study 40d before parturition after which 1 udder half was milked once daily (1×) and the other 4 times daily (4×) Mammary biopsies were collected at 15, 60, 120, and 230d of lactation. Average milk yield from the 4× side was 3 times higher than from the 1× side. Analysis of milk composition revealed that protein, lactose, and solids-not-fat percentages were lower in 1× than 4× udder halves. Mammary cell apoptosis was not affected by milking frequency. Mammary cell mitochondrial number, as estimated by succinate dehydrogenase staining, was higher in early lactation, decreasing as days in milk increased, and with increased milking frequency. Although mammary expression of α-lactalbumin (LALBA) and ß-casein (CSN2) was significantly increased in 4× glands, the expression of suppressors of cytokine signaling were similar between 1×- and 4×-milked halves. These results support the conclusion that changes in milk production in response to extreme differences in milking frequency may be related to alterations in mitochondrial number and lactose synthesis, but not apoptosis.

In silico QTL mapping of maternal nurturing ability with the mouse diversity panel.

Significant variation exists for maternal nurturing ability in inbred mice. Although classical mapping approaches have identified quantitative trait loci (QTL) that may account for this variation, the underlying genes are unknown. In this study, lactation performance data among the mouse diversity panel were used to map genomic regions associated with this variation. Females from each of 32 inbred strains (n = 8-19 dams/strain) were studied during the first 8 days of lactation by allowing them to raise weight- and size-normalized cross-foster litters (10 pups/litter). Average daily weight gain (ADG) of litters served as the primary indicator of milk production. The number of pups successfully reared to 8 days (PNUM8) also served as a related indicator of maternal performance. Initial haplotype association analysis using a Bonferroni-corrected, genome-wide threshold revealed 10 and 15 associations encompassing 11 and 13 genes for ADG and PNUM8, respectively. The most significant of these associated haplotype blocks were found on MMU 8, 11, and 19 and contained the genes Nr3c2, Egfr, Sec61g, and Gnaq. Lastly, two haplotype blocks on MMU9 were detected in association with PNUM8. These overlapped with the previously described maternal performance QTL, Neogq1. These results suggest that the application of in silico QTL mapping is a useful tool in discovering the presence of novel candidate genes involved in determining lactation capacity in mice.

Technical note: assessing the functional capacity of mitochondria isolated from lactating mammary tissue: choose your chelating agent wisely.

Previous work has indicated that respiratory activity of mitochondrial preparations prepared from lactating mammary tissue is often much lower than that of mitochondria isolated from other organs such as the liver. Initial studies in our own laboratory also found that mammary mitochondria prepared from lactating mice had much lower ATP synthesis activity than those isolated from liver tissue obtained from the same animals. In this paper, we describe an improved procedure for obtaining coupled mitochondria from the mammary tissue of lactating mice. Using a high-throughput assay for mitochondrial ATP synthesis, we demonstrated that mammary mitochondria, unlike liver mitochondria, are sensitive to the concentration of bovine serum albumin and to the choice of chelating agent used in the preparation and assay buffers. Mammary mitochondria prepared and assayed in buffers containing 1 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N' tetraacetic acid (EGTA) and 0.4% bovine serum albumin have a similar ATP synthesis activity as liver mitochondria. In addition, we show that the chelating agent EDTA ablates the ATP synthesis capacity of mammary mitochondria through a mechanism that does not involve the release of cytochrome c. We also demonstrate that these improved isolation and assay procedures are both scalable and applicable to bovine mammary tissue, and we describe optimal conditions for cryopreservation and recovery of functionally active mitochondria. This work will facilitate future studies aimed at determining the importance of mammary mitochondria to milk production.

Milk glucosidase activity enables suckled pup starch digestion.

UNLABELLED: ᅟ: Starch requires six enzymes for digestion to free glucose: two amylases (salivary and pancreatic) and four mucosal maltase activities; sucrase-isomaltase and maltase-glucoamylase. All are deficient in suckling rodents. OBJECTIVE: The objective of this study is to test (13)C-starch digestion before weaning by measuring enrichment of blood (13)C-glucose in maltase-glucoamylase-null and wild-type mice. METHODS: Maltase-glucoamylase gene was ablated at the N-terminal. Dams were fed low (13)C-diet and litters kept on low (13)C-diet. Pups were weaned at 21 days. Digestion was tested at 13 and 25 days by intragastric feeding of amylase predigested (13)C-α-limit dextrins. Blood (13)C-glucose enrichment was measured by gas chromatography combustion isotope ratio mass spectrometry (GCRMS) using penta-acetate derivatives. RESULTS: Four hours after feeding, blood (13)C-glucose was enriched by 26 × 10(3) in null and 18 × 10(3) in wild-type mice at 13 days and 0.3 × 10(3) and 0.2 × 103 at 25 days (vs. fasting p = 0.045 and p = 0.045). By jejunal enzyme assay, immunohistochemistry, or Western blots, there was no maltase activity or brush border staining with maltase-glucoamylase antibodies at 13 days, but these were fully developed in the wild-type mice by 25 days. In 13-day null mice, luminal contents were stained by maltase-glucoamylase antibodies. Lactating the mammary gland revealed maltase-glucoamylase antibody staining of alveolar cells. Reverse transcription/polymerase chain reaction (RT/PCR) of lactating glands revealed a secreted form of maltase-glucoamylase. CONCLUSIONS: (1) (13)C-α-limit dextrins were rapidly digested to (13)C-glucose in 13-day mice independent of maltase-glucoamylase genotype or mucosal maltase activity. (2) This experiment demonstrates that a soluble maltase activity is secreted in mouse mother's milk which enables suckling pup starch digestion well before brush border enzyme development. (3) This experiment with (13)C-α-limit dextrins needs to be repeated in human breast fed infants.

Cooperative interaction between mutant p53 and des(1-3)IGF-I accelerates mammary tumorigenesis.

Mammary tumorigenesis was analysed in transgenic mice which overexpress des(1-3)hIGF-I (WAP-DES) and/or a mutant form of p53 (p53172R-H). Nonlactating, multiparous WAP-DES mice exhibited hyperplastic lesions termed mammary interepithelial neoplasia (MIN) which constitutively expressed WAP-DES. By 23 months of age, 53% of the WAP-DES mice developed mammary adenocarcinomas. A 75% reduction in both apoptosis and proliferation was observed in the normal mammary glands of WAP-DES mice. Mammary tumor incidence in WAP-DES/p53 bitransgenic mice was similar to that of WAP-DES and 2 - 3-fold greater than that of nontransgenic and p53172R-H females. Tumor latency, however, was reduced by 8 months in bitransgenic mice as compared to mice of the other three genotypes. Aneuploidy was frequently observed in tumors from bitransgenic and p53172R-H mice, but not from mice expressing only the WAP-DES transgene. Expression of IGFBP3 was elevated in tumors from WAP-DES, but not bitransgenic mice, indicating an alteration in the p53/IGF-I axis. These studies indicate that overexpression of des(1-3)hIGF-I increases the frequency of MIN and stochastic mammary tumors and that the appearance of tumors displaying genomic instability is accelerated by mutant p53172R-H. Oncogene (2000) 19, 889 - 898.

Targeted disruption of the IGF-I receptor gene decreases cellular proliferation in mammary terminal end buds.

IGF-I mediates mammary ductal development through stimulation of terminal end bud (TEB) development; however, no published data exist on the mechanism through which this occurs. The mechanism of IGF-I action on the TEB was studied by determining the requirement for the IGF-I receptor (IGF-IR) in IGF-I-dependent ductal development. We hypothesized that loss of the IGF-IR would disrupt mammary ductal development through a combination of decreased proliferation or increased apoptosis. Because IGF-IR null mice die at birth, embryonic mammary gland transplantation was used to study the effects of a disrupted IGF-IR gene. Analyses of grafts after 4 or 8 wk of development demonstrated a limited growth potential of the null mammary epithelium in virgin hosts. Bromodeoxyuridine labeling and terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling showed that cell proliferation was significantly decreased in null TEBs, but apoptosis was not. In addition, both the size and number of TEBs were reduced in null outgrowths. In pregnant hosts, null ductal growth was stimulated beyond the level seen in virgin hosts. These findings directly establish a proliferation-dependent role for the IGF-IR in the cells of the TEB. Additionally, this study indicates that pregnancy-dependent compensatory mechanisms can stimulate mammary development in the absence of an IGF-IR.

Characterization of the change in type I and II insulin-like growth factor receptors of bovine mammary tissue during the pre- and postpartum periods.

Type I and II receptors for the insulin-like growth factors (IGF) were characterized in microsomes from bovine mammary tissue. Specific binding of [125I]IGF-I increased linearly with increasing microsomal protein concentrations. In contrast, IGF-II binding showed a curvilinear relationship over the concentration range tested, with a maximum at 120 micrograms/ml. Kinetic studies conducted at 4 C showed the binding reactions to be reversible. Competition studies showed recombinant human IGF-I (rh-IGF-I) to be 8-, 18-, and 1000-fold more potent at inhibiting [125I]rh-IGF-I binding than recombinant bovine IGF-II (rb-IGF-II), multiplication-stimulating activity, and insulin, respectively. rbIGF-II was 1.8- and 6-fold more potent at inhibiting [125I]rbIGF-II binding than rhIGF-II and multiplication-stimulating activity. Specific binding of [125I]IGF-I and -II was measured in microsomes prepared from cows (n = 47) ranging from 138 days prepartum to 411 days postpartum. IGF-I binding declined during the prepartum period, increased 75% with the onset of lactation, and then declined during the postpartum period. Scatchard analysis showed the presence of a single class of high affinity binding sites for both ligands, with type II receptors being about 10-fold more prevalent than type I receptors. The survey and Scatchard data support the conclusion that the onset of lactation coincides with an increase in the number of type I receptors present in mammary tissue. This increase supports the contention that IGF-I may play an important role in modulating the metabolic activity of the bovine mammary gland.

Perinatal expression of type I IGF receptors in porcine small intestine.

The gastrointestinal tract of the pig undergoes enhanced growth as well as morphological and functional differentiation during the perinatal period. Concurrently, porcine neonates ingest physiologically significant amounts of insulin-like growth factor-I (IGF-I) via colostrum and milk. The objectives of this study were to examine newborn pig small intestine for the presence of high affinity, IGF-I receptors and to evaluate the possible contributions of maternally derived and locally produced IGF-I to receptor-mediated postnatal growth of intestine. The specific binding of 125I-IGF-I to membranes prepared from scraped intestinal mucosa was time, temperature, and pH dependent; optimal conditions were 48 h, 4 C, and a pH of 7.8, respectively. Several pure peptides were evaluated for competition with 125I-IGF-I in binding to intestinal membrane sites. The relative order of competition was IGF-I greater than insulin-like growth factor-II (IGF-II) greater than insulin, whereas bombesin and epidermal growth factor were noncompetitive. Chemical cross-linking of 125I-IGF-I to binding sites, followed by denaturing SDS-PAGE and autoradiography, demonstrated labeled protein complexes of Mrs 135,000 and 260,000. Both autoradiographic bands were diminished when excess unlabeled IGF-I or IGF-II was included in the binding step. Insulin at higher concentrations also slightly inhibited labeling of both membrane proteins. Membranes prepared from intestinal mucosa of piglets at days 0 (less than 2-h old, colostrum-deprived), 3, 5, and 21 postnatal were evaluated for developmental variations in specific binding of 125I-IGF-I. Binding was highest at birth, declined (-43%) by day 3, remained low at day 5, and increased by day 21. Receptor affinity was relatively invariant whereas receptor number (per mg membrane protein) was variable. Intestine wt increased disproportionately to body wt between days 0 to 3, postnatal. Radioimmunoassay of extracts of the corresponding intestinal mucosa revealed a significant increase in content of IGF-I by day 3 (P = 0.05), whereas RNA dot-blot hybridization demonstrated low and unchanging IGF-I mRNA abundance in intestine. The quantitative variations in IGF-I protein content and IGF receptor numbers temporally coincide with intestinal villous growth, cessation of intestinal transport of macromolecules (closure), and onset of maturation of intestinal function.

Targeted expression of des(1-3) human insulin-like growth factor I in transgenic mice influences mammary gland development and IGF-binding protein expression.

To test the hypothesis that insulin-like growth factor I (IGF-I) regulates mammary gland development and lactation, the expression of both human (h) IGF-I and des(1-3)hIGF-I was targeted to the mammary gland in transgenic mice using a novel exon replacement strategy and the rat whey acidic protein (rWAP) gene regulatory sequences. Both transgenes expressed a 0.7-kilobase messenger RNA (mRNA). The abundance of WAP-IGE-I and WAP-DES mRNA on day 10 of lactation ranged from 0.2-1.0% and 0.2-13% of the endogenous mouse WAP mRNA, respectively. For WAP-DES mice, transgene expression was greatest from midpregnancy throughout lactation. Western blot analysis showed the presence of correctly processed hIGF-I in milk from these transgenic mice. This hIGF-I was capable of stimulating protein synthesis in cultured rat L6 myoblasts. Ligand blotting indicated changes in mammary gland secretion of IGFBP in response to WAP-DES expression. Histological analysis of mammary tissue from mice overexpressing des(1-3)hIGF-I showed incomplete mammary involution, ductile hypertrophy, and loss of secretory lobules associated with increased deposition of collagen. These changes are believed to occur through autocrine and paracrine effects of des(1-3)-hIGF-I on both epithelial and stromal cells.

Inability of overexpressed des(1-3)human insulin-like growth factor I (IGF-I) to inhibit forced mammary gland involution is associated with decreased expression of IGF signaling molecules.

Overexpression of des(1-3) human insulin-like growth factor I (IGF-I) in the mammary glands of transgenic mice (WAP-DES) inhibits apoptosis during natural, but not forced, mammary involution. We hypothesized that this differential response would correlate with the expression of IGF signal transducers. Forced and natural involution were analyzed in nontransgenic and WAP-DES mice beginning on day 16 postpartum. During natural involution, mammary gland wet weight was higher and apoptosis was lower in WAP-DES than in nontransgenic mice. The WAP-DES transgene had no effect on these parameters during forced involution. Mammary tissue concentrations of the transgene protein were 2- to 10-fold higher than those of endogenous IGF-I. Western blot analysis of pooled mammary tissue extracts demonstrated only slightly higher phosphorylation of the IGF signal transducers insulin receptor substrate-1 (IRS-1) and Akt in the WAP-DES than in nontransgenic mice. Dramatic early reductions in phospho-IRS-1, phospho-Akt, IRS-1, IRS-2, and Akt proteins occurred during forced, but not natural, involution. The abundance of the IGF-I receptor and the messenger RNAs for the IGF-I receptors, IRS-1 and -2, were not affected by either genotype or involution. These findings support the conclusions that mammary cells lose their responsiveness to insulin-like signals during forced involution, and that posttranscriptional or posttranslational regulation of IRS-1 and IRS-2 may play a role in this loss.

Developmental and hormonal signals dramatically alter the localization and abundance of insulin receptor substrate proteins in the mammary gland.

Insulin receptor substrates (IRS) are central integrators of hormone, cytokine, and growth factor signaling. IRS proteins can be phosphorylated by a number of signaling pathways critical to normal mammary gland development. Studies in transgenic mice that overexpress IGF-I in the mammary gland suggested that IRS expression is important in the regulation of normal postlactational mammary involution. The goal of these studies was to examine IRS expression in the mouse mammary gland and determine the importance of IRS-1 to mammary development in the virgin mouse. IRS-1 and -2 show distinct patterns of protein expression in the virgin mouse mammary gland, and protein abundance is dramatically increased during pregnancy and lactation, but rapidly lost during involution. Consistent with hormone regulation, IRS-1 protein levels are reduced by ovariectomy, induced by combined treatment with estrogen and progesterone, and vary considerably throughout the estrous cycle. These changes occur without similar changes in mRNA levels, suggesting posttranscriptional control. Mammary glands from IRS-1 null mice have smaller fat pads than wild-type controls, but this reduction is proportional to the overall reduction in body size. Development of the mammary duct (terminal endbuds and branch points) is not altered by the loss of IRS-1, and pregnancy-induced proliferation is not changed. These data indicate that IRS undergo complex developmental and hormonal regulation in the mammary gland, and that IRS-1 is more likely to regulate mammary function in lactating mice than in virgin or pregnant mice.

Effects of elevated blood insulin-like growth factor-I (IGF-I) concentration upon IGF-I in bovine mammary secretions during the colostrum phase.

The objectives of these studies were to determine if the concentration of insulin-like growth factor-I (IGF-I) in mammary colostrum secretions could be altered through manipulation of IGF-I concentrations in blood and to compare the temporal changes of IGF-I in mammary secretions to those occurring for IgG1. Milking of 15 pregnant Holstein cows was stopped at 8 weeks prepartum and they were randomly assigned to one of three treatments. A control (C) treatment consisted of feeding the animals 100% of NRC requirements for protein and energy. A second group of cows was fed as the control group and injected with 1.8 mumol bovine GH/day. The third group was fed at 70% of NRC requirements for protein and energy to cause a moderate nutrient restriction (NR). Body weight was measured weekly. Blood was collected by tail venepuncture at 4 h intervals for 24 h. Mammary secretions were collected and pooled among contralateral front and rear quarters (diagonal) for measurement of volume, IGF-I and IgG1 concentrations. Samples were collected at -7, -5, -2, 0 and 1 week postpartum. Cows on the NR treatment failed to gain weight during the dry period compared with C cows (P < 0.05). Blood GH and IGF-I concentrations (P > 0.1) were unaffected by NR treatment. Cows treated with GH had higher (P < 0.01) serum GH and IGF-I levels throughout the entire treatment period, and higher serum IgG1 at 5 and 2 weeks prepartum (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cell apoptosis by insulin-like growth factor I.

Correct temporal and spatial regulation of apoptosis is critical for normal mammary gland development and lactation. Previous work with a strain of transgenic mice that overexpress des(1-3)hIGF-I during pregnancy and lactation suggested that this growth factor inhibits apoptosis. The hypothesis tested within these studies is that overexpression of des(1-3)hIGF-I within the mammary gland inhibits apoptosis and the expression of apoptosis-associated genes that are known to be activated by the transcription factor AP-1. This inhibition of apoptosis was further posited to predispose the tissue to carcinogenesis. TUNEL analysis of mammary tissue from transgenic mice that overexpress des(1-3)hIGF-I under control of the rat whey acidic protein promoter showed only 25% (P < 0.05) of the number of apoptotic cells found in nontransgenic mice at the same stage of lactation. Northern analysis of RNA from these animals showed a 75% (P = 0.08) reduction in c-Jun mRNA abundance. Histological analysis of mammary tissue from nonlactating multiparous WAP-DES mice ranging in age from 13 to 25 months showed a variety of hyperplastic lesions. These lesions aberrantly expressed the transgene. At 23 months of age 50% of the transgenic mice within this study developed adenocarcinomas. These results support the conclusion that inhibition of apoptosis within the mammary gland by IGF-I involves decreased activity of AP-1 and predisposes the tissue to tumors.

Effects of dietary insulin-like growth factor I on growth and insulin-like growth factor receptors in neonatal calf intestine.

Colostrum is rich in IGF-I and IGF-II, and the dietary effects of recombinant human (rh)IGF-I on the newborn are of interest. The objective of this study was to examine the effects of dietary rhIGF-I on intestinal tissue growth and populations of IGF receptors. Twenty-three male diary calves were fed one of three experimental diets: 1) milk replacer plus isolated colostrum-derived globulins (MR-), 2) same as 1 plus 750 ng of rhIGF-I/mL (MR+), or 3) pooled cow colostrum (COL). After the first four feedings, all calves received milk replacer without additional globulins; calves fed the MR+ diet continued to receive the addition of 750 ng of rhIGF-I/mL until the experiment ended at 7 d after birth. Calves were killed and intestinal tissue was collected for in vitro [3H]thymidine incorporation studies. Incorporation differed among intestinal regions (duodenum, jejunum, and ileum). The MR+ calves had greater (P < .01) [3H]thymidine incorporation per unit of DNA than either the COL or MR- calves (31.8 vs 18.6 and 11.5 x 10(3) dpm/microgram of DNA, respectively). Competitive binding analysis indicated the presence of specific type 1 and type 2 intestinal IGF receptors. The IGF-I was more potent than IGF-II and insulin at inhibiting [125I]rhIGF-I binding (ED50 was 1.84, 9.17, and 1.91 ng/mL, respectively). The IGF-II was the only ligand capable of inhibiting [125I]rhIGF-II binding (ED50 was .30 nmol/mL).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor (IGF)-I and -II and IGF binding proteins in serum and mammary secretions during the dry period and early lactation in dairy cows.

Concentrations of IGF-I and IGF-II, and IGF binding proteins (IGFBP) in serum and mammary gland secretions were surveyed during the dry period and early lactation of 30 Holstein cows. Although there was a threefold drop in the concentration of IGF-I in serum from the last week of the dry period to parturition (81 +/- 7 to 24 +/- 3 ng/ml, P less than .01), there was no significant change in serum IGF-II concentration during this period (150 +/- 17 vs 173 +/- 13 ng/ml, P greater than .05). Furthermore, a 57% increase in serum IGF-I was observed from the last week of lactation to the second week of drying off (100 +/- 5 to 157 +/- 8 ng/ml, P less than .05). Changes in serum IGF-II were not observed (126 +/- 11 vs 150 +/- 10 ng/ml, respectively; P greater than .05). Although IGF-I, IGF-II, and IGFBP concentrations in mammary secretions peaked 2 wk before parturition (2.95 +/- 1.1, 1.83 +/- .6, and 7.27 +/- .76 micrograms/ml, respectively), total output/quarter was highest in colostrum (394 +/- 119, 295 +/- 132, and 2,680 +/- 1,967 micrograms/quarter, respectively). Weekly milking of two individual quarters during the dry period did not affect (P greater than .05) IGF-I or IGF-II concentration (ng/ml) or total output (microgram/quarter) and milk yield in colostrum and milk (2 wk and 7 wk) compared with the ipsilateral quarter. The data support the hypothesis that IGF-I may be transported by the mammary gland epithelium. Furthermore, the secretion mechanisms of IGF-I, IGF-II, and IGFBP by the gland may be related to each other.

Enforced epithelial expression of IGF-1 causes hyperplastic prostate growth while negative selection is requisite for spontaneous metastogenesis.

The insulin-like growth factor-1 (IGF-1) signaling axis is important for cell growth, differentiation and survival and increased serum IGF is a risk factor for prostate and other cancers. To study IGF-1 action on the prostate, we created transgenic (PB-Des) mice that specifically express human IGF-1(des) in prostate epithelial cells. This encodes a mature isoform of IGF-1 with decreased affinity for IGF binding proteins (IGFBP) due to a 3-amino acid deletion in the N terminus. Expression of IGF-1(des) was sufficient to cause hyperplastic lesions in all mice, however the well-differentiated lesions did not progress to adenocarcinoma within a year. Remarkably, crossing the PB-Des mice to an established model of prostate cancer delayed progression of organ-confined tumors and emergence of metastatic lesions in young mice. While dissemination of metastatic lesions was widespread in old bigenic mice we did not detect IGF-1(des) in poorly differentiated primary tumors or metastatic lesions. Expression of endogenous IGF-1 and levels of P-Akt and P-Erk were reduced independent of age. These data suggest that increased physiologic levels of IGF-1 facilitate the emergence of hyperplastic lesions while imposing a strong IGF-1-dependent differentiation block. Selection against IGF-1 action appears requisite for progression of localized disease and metastogenesis.

Chickpeas as a protein and energy supplement for high producing dairy cows.

Thirty lactating Holstein cows were randomly assigned to one of three diets to evaluate chickpeas as dietary supplement from wk 4 to 16 postpartum. Diets contained chickpeas at 0, 50, and 100% of the concentrate DM in place of corn and soybean meal. Total mixed diets, fed individually, contained concentrate, corn silage, and alfalfa hay at 52, 32, and 16% of DM. Milk yield (34.5, 35.1, 35.7 kg/d) was higher for cows fed 100% chickpeas than 0% chickpeas. Milk medium-chain (C12 to C16) fatty acids (53.8, 48.6, 45.5 g/100 g fat), milk long-chain (C18) fatty acids (29.4, 35.0, 38.8 g/100 g fat), and milk protein (3.20, 3.09, 2.96%) differed for all three diets. Ruminal acetate: propionate (2.0, 2.0, 2.4) was highest for cows fed 100% chickpeas. Total essential amino acid concentration in blood serum (105.3, 95.9, 89.4 mumol/dl) was higher for cows fed 0% chickpeas than 100% chickpeas. Lactational responses are discussed with respect to increased intake of fat, increased degradability of dietary protein, and decreased plasma essential amino acids.

IGF and insulin action in the mammary gland: lessons from transgenic and knockout models.

Transgenic and knockout mice have become valuable experimental systems with which to study specific molecular events within the mammary gland of an intact animal. These models have provided a wealth of information about the effects of a number of oncogenes and growth factors. This review focuses on results obtained from the application of transgenic and knockout models to determine the roles of insulin and insulin-like growth factors (IGF) in the regulation of mammary gland development, lactation and tumorigenesis. Transgenic models which overexpress IGF-I or -II display specific alterations in mammary gland development and an increased incidence of mammary tumors. Analysis of mammary gland development in knockout mice which are deficient in IGF-I or the IGF-I receptor supports the conclusion that the IGF system is important for normal mammary gland development. This review discusses these observations in detail and attempts to fit them into a larger picture of IGF and insulin action in the mammary gland.

Genetic manipulation of the IGF-I axis to regulate mammary gland development and function.

Insulin-like growth factor I (IGF-I) is known to regulate mammary gland development. This regulation occurs through effects on both cell cycle progression and apoptosis. Our laboratory has studied the IGF-I-dependent regulation of these processes by using transgenic and knockout mouse models that exhibit alterations in the IGF-I axis. Our studies of transgenic mice that overexpress IGF-I during pregnancy and lactation have demonstrated that this growth factor slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation but has minimal effects during early lactation on milk composition or lactational capacity. In contrast, our analysis of early developmental processes in mammary tissue from mice carrying a targeted mutation in the IGF-I receptor gene suggests that IGF-dependent stimulation of cell cycle progression is more important to early mammary gland development than potential anti-apoptotic effects. With both models, the effects of perturbing the IGF-I axis are dependent on the physiological state of the animal. The diminished ductal development that occurs in response to loss of the IGF-I receptor is dramatically restored during pregnancy, whereas the ability of overexpressed IGF-I to protect mammary cells from apoptosis does not occur if the mammary gland is induced to undergo forced involution. Data from our laboratory on the expression of IGF-signaling molecules in the mammary gland suggest that this effect of physiological context may be related to the expression of members of the insulin receptor substrate family.