Monocarboxylate transporter genes in the mammary gland of lactating cows

This study is the first to examine the expression of the 14 monocarboxylate transporter genes (MCT1–MCT14) in the mammary gland of mammals. RT-PCR, Western blot, immunohistochemistry, and immunofluorescence confocal laser microscopy were applied in a comprehensive approach to assess the expression and cellular localization of MCTs in the mammary gland of lactating cattle. RT-PCR revealed the existence of nine MCT isoforms, namely MCT1, MCT2, MCT3, MCT4, MCT5, MCT8, MCT10, MCT13, and MCT14 in cow mammary gland. The amplified cDNA segments were confirmed by sequence analysis and deposited in the GenBank. Using the commercially available antibodies against MCT1–MCT8, Western blotting verified the protein expression of MCT1, MCT2, MCT3, MCT4, MCT5, and MCT8 in the cow mammary gland. The precise cellular localization of the identified MCT proteins showed that both MCT1 and MCT2 were basolaterally localized on the cow mammary alveolar epithelial cells. In contrast, MCT4 protein signal was expressed on the apical membrane of these alveolar epithelia. MCT8, however, was predominantly localized on the basolateral membranes of the lactocytes, along with its weak labeling on the apical membrane of the same cells. No immunoreactive staining for MCT3 and MCT5 proteins could be detected histochemically in lactating bovine mammary tissue. Additionally, we proved the colocalization of CD147 with both MCT1 and MCT4 on the boundaries of the cow mammary alveolar epithelia. The existence and localization pattern of MCT genes in the mammary gland of lactating cows suggest their possible involvement in the transport of essential elements required for milk synthesis and secretion.     

Isolation and characterization of fatty acid binding proteins from mammary tissue of lactating rats.

A protein fraction with fatty acid binding activity has been isolated from mammary tissue from lactating rats by a process involving DEAE-cellulose ion-exchange chromatography, heat treatment, CM-cellulose ion-exchange chromatography and finally ammonium sulphate precipitation. The purified fraction migrated as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 14400. However, when this protein fraction was electrophoresed under non-dissociating conditions, two species were observed in a 4:1 ratio. The two components were separated using h.p.l.c. Both bind fatty acids and appear to have similar amino acid compositions although exhibiting different pI values of 4.8 and 4.9. The mammary fatty acid binding proteins appear to be very similar to the fatty acid binding protein isolated from rat heart based on the electrophoretic mobilities and amino acid composition. The major mammary form (pI 4.9) has been partially sequenced and the amino acid sequences obtained can be aligned with 67 residues of the revised rat heart amino acid sequence [Heuckeroth, Birkenmeier, Levin & Gordon (1987) J. Biol. Chem. 262, 9709-9717]. Both mammary species also showed immunochemical identity to rat heart fatty acid binding protein when tested with an anti-serum raised against the heart protein. Anti-sera raised against the minor mammary form (pI 4.8) specifically precipitated this form under non-denaturing conditions but both forms after they had been denatured. Quantitative immunoassays using the anti-(heart fatty acid binding protein) serum showed that concentrations of the fatty acid binding proteins present in mammary cytosols increase during lactation and increase further after feeding a high-fat diet.     

Whole-body protein metabolism in lactating and nonlactating women

The adaptive responses of body protein metabolism to lactation were characterized in women at 1, 5, and 12 mo postpartum and in nulliparous controls during a controlled diet of measured protein and energy intakes by nitrogen balance, a constant infusion of [13C]bicarbonate, and a primed constant infusion of [1-13C]leucine and [alpha-15N]-lysine. Dietary energy intakes in the lactating women were 27% greater than those in the nulliparous controls. Despite these differences, lactating women had significantly lower nitrogen balances compared with the nonlactating women (-4.0 +/- 37.8 vs. +44.7 +/- 30.8 mg.kg-1.day-1). No significant differences in amino acid flux, oxidation, or incorporation into protein were detected during fasting conditions in the two groups of women. However, significantly positive associations were noted between dietary intakes and the variables of protein metabolism in the lactating women. A more complete understanding of the mechanisms that regulate the disposition of dietary nutrients into maternal body stores or milk production will enhance the determination of nutrient requirements in lactating women.     

Oxytocin binding sites in lactating rabbit mammary gland.

Material which specifically binds oxytocin was prepared from a crude preparation of lactating rabbit mammary gland by purification on a sucrose density gradient. On examination of activities of enzyme markers and the molar ratio of cholesterol to phospholipid, this material was considered to be a highly purified plasma membrane fraction. For the determination of specificity and time course of oxytocin binding, a Scatchard plot analysis was carried out for the crude and purified fractions. Dissociation constant (Kd) and binding capacity values were found to be as follows: crude, Kd equals 1.83 X 10(-9) M, capacity equals 670 fmol/mg protein; purified, Kd equals 2.8 X 10(-9) M, capacity equals 1700 fmol/mg protein. Treatment of the purified material with different detergents resulted in loss of all [3H]oxytocin binding capacity. However, preincubation of this material with [3H]oxytocin prior to detergent treatment resulted in solubilization of a receptor-hormone complex. This complex remained in the supernatant even after centrifugation at 210 000 X g for 30 min. Using oxytocin analogs, we have shown this solubilized complex to be oxytocin specific.     

Effect of prepartum diet on postpartum ovarian activity in Holstein cows in a pasture-based dairy system

The hypothesis was that supplementation during the late prepartum period will differentially affect reproductive and productive variables according to parity. Primiparous (n=22) and multiparous (n=22) pregnant autumn calving Holstein cows were stratified in two groups according to parity (primiparous or multiparous) and within each group were randomly assigned to two treatments: (a) low supplemented (LS) or (b) high supplemented (HS) prepartum diet. The LS group was offered 5.2 kg/cow/day (DM basis) of wheat silage, and the HS group 4.7 kg cow/day (DM basis)/of corn silage and 3.6 kg (DM basis) of wheat bran+12 g of urea. Both groups grazed on natural pastures. After calving, all cows received the same diet. The experimental period was from 3 weeks before calving to 7 weeks postpartum (PP); body condition score (BCS) and blood samples for hormonal analyses were obtained weekly and ovarian ultrasonography was conducted three times per week. The loss in BCS around calving was less pronounced in HS cows, but only multiparous supplemented cows maintained BCS throughout the study. Non-esterified fatty acids (NEFA) increased during the prepartum period in the LS but not in the HS cows, with peak values occurring on day 14 PP in all groups. During the remainder of the experiment NEFA was greater in LS than in HS cows. Prepartum treatment did not affect the proportion of cows that had ovulations from the first dominant follicle postpartum, but decreased the interval to first ovulation in multiparous cows (22.9 compared with 38.2 days; P<0.05). This was associated with greater plasma IGF-I concentrations at the time the dominant follicle of the first follicular wave reached its maximum diameter (8.0 compared with 3.6 nmol/L; P<0.05). However, prepartum treatment had no effect on onset of ovarian activity in primiparous cows. Supplementation had no effect on milk production or milk protein percentage but increased milk fat percentage. We conclude that feeding a high-supplemented prepartum diet to multiparous cows allowed them to maintain BCS around calving, and this was associated with greater concentrations of IGF-I and an earlier onset of estrous cycles after calving.     

Comparison of alkaline phosphatase activity in normal, lactating and neoplastic rat mammary tissue

1. Alkaline phosphatase activity in NMU-induced rat mammary tumours was compared with activity in normal and lactating mammary gland. 2. Both tumour and normal mammary alkaline phosphatase were sensitive to heat inactivation and inhibition by phenylalanine. 3. Specific activity of enzyme in tumours was comparable to normal mammary tissue. 4. Mammary gland alkaline phosphatase increased markedly in late pregnancy and early lactation. 5. Bromocryptine treatment had no effect on enzyme activity in lactating mammary gland.     

Efflux of chloride from lactating rat mammary tissue slices

1. The efflux of chloride (using 36Cl) from lactating rat mammary tissue slices has been investigated. 2. Chloride efflux was found to be temperature dependent; lowering the temperature of the incubation medium reduced the fractional efflux. 3. The stilbene derivatives DIDS was without effect on the fractional release of Cl when studied at 20 degrees C. However, DIDS was found to attenuate the increase in efflux found upon transferring the tissue from a medium maintained at 4 degrees C to one at 20 degrees C. 4. The loop-diuretic furosemide, also reduced the temperature-sensitive portion of Cl efflux. 5. Chloride efflux was transiently increased when tissue slices were transferred from a medium containing gluconate as the principal anion to one containing Cl. 6. The results appear to confirm that mammary Cl transport is mediated via anion exchange and via (Na + K + Cl) cotransport.     

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 K_m of 20 μM and a V_max of 386 μmol/kg cell water/30 min. Substrate specificity studies indicated a strong interaction of β-amino acids with the taurine transport system. Taurine transport in lactating sow mammary tissue is therefore a high affinity, sodium-dependent mechanism specific for β-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.     

Risk factors for resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows

The objectives of this study were to evaluate factors associated with resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows. Holstein cows, 6396 from four dairy farms were evaluated to determine the relationships among parity, body condition score (BCS) at calving and at AI, season of year when cows calved, and milk yield on resumption of postpartum estrous cycles by 65 days postpartum, and all the previous variables, estrual or anestrus and AI protocol on conception rates and embryonic survival at the first postpartum insemination. Cows had their estrous cycle pre-synchronized with two PGF_2α injections given 14 days apart and were inseminated between 69 and 82 days postpartum following either an estrous or ovulation synchronization protocol initiated 12–14 days after the presynchronization. Blood was sampled and analyzed for progesterone twice, 12–14 days apart, to determine whether cows had initiated onset of estrous cycles after calving. Cows were scored for body condition in the week after calving, and again at AI, between 69 and 82 days postpartum. Pregnancy was diagnosed at 30 ± 3 and 58 ± 3 days after AI. Farm influenced all reproductive outcomes evaluated. More (P < 0.0001) multiparous than primiparous cows had initiated estrous cycles. Onset of estrous cycles was also influenced (P < 0.01) by BCS at calving and at AI, BCS change, season, and milk yield. More (P < 0.001) cows that had initiated estrous cycles than anestrous cows were pregnant at 30 and 58 days after AI, but anestrus did not affect pregnancy loss. Conception rates were also influenced (P < 0.01) by parity, BCS at calving and AI, BCS change, and season; however, milk yield and insemination protocol were not associated with conception rates at 30 and 58 days after AI. Factors that reduced conception rate on day 30 after AI also increased pregnancy loss between 30 and 58 days of gestation. Improving BCS at calving and AI, minimizing losses of BCS after calving, and hastening onset of estrous cycles early postpartum are all expected to increase conception because of enhanced embryonic survival.     

Relationships between calf birth weight, prepartum concentrations of plasma energy metabolites and resumption of ovulation postpartum in Limousine suckled beef cows

The aim of this study was to determine the relationship between energy status before calving and calf birth weight and their potential effects on interval between calving and first ovulation. Sixty-nine Limousine, suckled beef cows were sampled weekly over a 3-yr period during the last 2 m.o. of pregnancy to determine the concentrations of nonesterified fatty acids (NEFA), beta-3-hydroxybutyrate (beta-OHB), glucose and glycerol. After parturition, progesterone concentrations were measured weekly to determine time of resumption of ovulation. Cows were allotted to 3 groups according to calf birth weight (Heavy: > 44 kg, n = 37; Medium: 39 to 43 kg, n = 56; and Light: < 38 kg, n = 45) and to postpartum ovarian resumption of cyclicity (Late: > 11 wk, n = 41; Mid: 7 to 10 wk, n = 57; and Early: < 6 wk, n = 40). Puerperium glycaemia of the dams was steady state (0.66 +/- 0.03 g/L) and was not related to calf birth weight. Plasma NEFA, beta-OHB and glycerol values were higher (P < 0.05) in Heavy than in Medium and Light group dams during the last 4 wk of pregnancy. Interval between calving and first ovulation was significantly longer for primiparous than for multiparous cows (respectively, 9.9 +/- 2.0 and 7.7 +/- 1.4 wk; P < 0.05). Calf birth weight was not related to time of first ovulation. Late primiparous cows had higher NEFA plasma concentrations than Mid and Early group primiparous cows during the last 4 wk of pregnancy, whereas NEFA plasma concentrations were not related to interval between calving and first ovulation in multiparous cows. Thus, lipomobilization increased with calf birth weight during the last 4 wk of pregnancy. High level of body reserves mobilization was associated with delayed first ovulation in primiparous but not in multiparous cows.     

Glutathione utilization by lactating bovine mammary secretory tissue in vitro.

gamma-Glutamyltransferase (D-glutamyl transpeptidase, EC 2.3.2.2) activity has been shown to be located predominantly on the extracellular surface of the plasma membrane of lactating bovine mammary cells. Radioactive label from both oxidized ([14C]-gamma-glutamyl) and reduced ([35S]cysteinyl) glutathione was taken up and incorporated into acid-precipitable proteins of mammary tissue. Uptake was shown to involve the transport of free amino acids, and incorporation was shown to involve the action of gamma-=glutamyltransferase. These results indicate that lactating mammary tissue utilizes the constituent amino acids of glutathione for milk-protein synthesis.     

Volume-activated K(+)(Rb(+)) efflux in lactating rat mammary tissue

The effect of cell swelling, induced by a hyposmotic shock, on K(+)(Rb(+)) efflux from lactating rat mammary tissue explants has been studied. A hyposmotic challenge increased the fractional release of K(+)(Rb(+)) from mammary tissue in the absence and presence of the loop-diuretic bumetanide (100 microM). However, the volume-sensitive moiety of K(+)(Rb(+)) efflux was proportionately larger when bumetanide was present in the incubation medium. On the other hand, a hyposmotic shock appeared to reduce the bumetanide-sensitive component of K(+)(Rb(+)) efflux. The increase in K(+)(Rb(+)) efflux, induced by cell swelling, was dependent upon the extent of the hyposmotic challenge. In the presence of bumetanide, substituting Cl(-) with NO(3)(-) reduced the initial increase in volume-sensitive K(+)(Rb(+)) efflux. However, volume-sensitive K(+)(Rb(+)) release was prolonged in the presence of NO(3)(-). Volume-activated K(+)(Rb(+)) efflux from rat mammary tissue explants was inhibited by quinine. Cell swelling increased the intracellular concentration of Ca(2+) in a fashion which depended on the presence of extracellular Ca(2+). However, removing extracellular Ca(2+) did not inhibit volume-activated K(+)(Rb(+)) efflux from rat mammary tissue explants. The results are consistent with the presence of volume-activated K(+) channels in lactating rat mammary tissue. Volume-activated K(+) efflux may play a central role in mammary cell volume regulation.     

Risk factors for postpartum ovarian cysts and their spontaneous recovery or persistence in lactating dairy cows

Cystic ovarian disease is a major cause of reproductive failure and economic loss for the dairy industry. Many cysts that develop during the early postpartum period regress spontaneously. However, it is difficult to decide at what point it would be more cost effective to treat ovarian cysts than to wait for spontaneous recovery. The objective of this study was to analyze risk factors for the development of the ovarian cystic condition during early and late postpartum, and for its persistence or recovery during the pre-service period in lactating dairy cows. Using multiple logistic regression, we analyzed data derived from 873 lactating dairy cows from a single herd. An ovarian cyst was diagnosed if it was possible to observe a single follicular structure with an antrum diameter > or = 25 mm in the absence of a corpus luteum in three sonograms performed at 7-day intervals. The cystic condition was denoted as early if the cyst was diagnosed 43-49 days postpartum, and late if detected 57-63-day postpartum. Spontaneous cyst regression before 60-day postpartum was regarded as early cystic recovery. For the early cystic group, there were no significant effects of lactation number, body condition score on prepartum Day 60, at parturition or on postpartum Day 30, or of body condition loss from parturition to 30-day postpartum. Cows calving in summer were 2.6 times more likely to develop ovarian cysts than those giving birth in winter. The risk of having a cyst was 1.9 times higher in cows with an abnormal puerperium. A 1-kg increase in milk yield raised the risk of cysts by a factor of 1.05. A 1-unit increase in body condition score (scale from 1 to 5) from prepartum Day 60 to parturition increased the risk of cyst development 8.4 times. Milk production and lactation number were negatively correlated with spontaneous early cyst recovery. A 1-kg decrease in milk production increased the probability of cyst recovery by a factor of 1.06, and a 1-unit drop in lactation number was associated with a 1.4-fold increased probability of cyst recovery. For the late cystic group, there were no significant effects of abnormal puerperium and body score data, except for a prepartum change in body score. Calving season (Odds ratio: 2.3), lactation number (Odds ratio: 1.36), increased milk production (Odds ratio: 1.05) and increased body condition score during the prepartum period (Odds ratio: 4.3) were all related to an increased risk of ovarian cysts. The probability of having a late cyst was 36.6 times greater in cows with early cysts. These findings suggest that it would be profitable to treat multiparous cows having cysts very early in the postpartum period, while treatment of primiparous cows should be delayed, at least until the end of the pre-service period, to provide the opportunity for spontaneous recovery.     

Regulation of protein synthesis in lactating rat mammary tissue by cell volume

The effect of changing cell volume on rat mammary protein synthesis has been examined. Cell swelling, induced by a hyposmotic challenge, markedly increased the incorporation of radiolabelled amino acids (leucine and methionine) into trichloroacetic acid (TCA)-precipitable material: reducing the osmolality by 47% increased leucine and methionine incorporation into mammary protein by 147 and 126% respectively. Conversely, cell shrinking, induced by a hyperosmotic shock, almost abolished the incorporation of radiolabelled amino acids into mammary protein: increasing the osmolality by 70% reduced leucine and methionine incorporation into mammary protein by 86 and 93% respectively. The effects of cell swelling and shrinking were fully reversible. Volume-sensitive mammary tissue protein synthesis was dependent upon the extent of the osmotic challenge. Isosmotic swelling of mammary tissue, using a buffer containing urea (160 mM), increased the incorporation of radiolabelled leucine into TCA-precipitable material by 106%. Swelling-induced mammary protein synthesis was dependent upon calcium: removing extracellular calcium together with the addition of EGTA markedly reduced volume-activated protein synthesis. Cell swelling-induced protein synthesis was inhibited by the Ca(2+) ATPase blocker thapsigargin suggesting that volume-sensitive protein synthesis is dependent upon luminal calcium.