Effect of photoperiod before and during first gestation on milk production and prolactin concentration in dairy heifers.

Holstein heifers (n = 45) were subjected to treatments according to a 2 × 2 factorial design where the main effects were the photoperiod treatments during the second isometric (ISO, 52-61 wk of age) and the second allometric (ALLO, 62 wk of age to 8 wk before calving) periods of mammary gland development. During the ISO period, heifers were subjected to either a short-day photoperiod (SDP; 8 h light, 16 h dark; n = 22) or a long-day photoperiod (LDP; 16 h light, 8 h dark; n = 23). During the ALLO period, the photoperiodic treatments were either maintained (SDP:SDP, n = 11; LDP:LDP, n = 11) or switched (SDP:LDP, n = 11; LDP:SDP, n = 12). The treatments ended 8 wk before calving. All animals were then subjected to about 16 h of light per day. Serum prolactin (PRL) concentration during the ISO period was greater in heifers exposed to LDP than in those exposed to SDP. For the first 20 wk of the ALLO period, heifers exposed to LDP had greater serum concentration of PRL than those exposed to SDP. On the other hand, previous exposure to LDP during the ISO period reduced the concentration of PRL compared with those exposed to SDP during that period. During the second 20 wk of the ALLO period, PRL concentration remained greater in the serum of heifers then exposed to LDP than SDP, but serum PRL was greater in heifers exposed to LDP during the ISO period. During the last weeks before calving, when all animals were exposed to LDP, previous exposure to LDP during the ALLO period reduced serum PRL. Early-lactation milk (wk 1-5) and energy-corrected milk (wk 2-6) production were higher in the heifers exposed to SDP than in those exposed to LDP during ALLO. Photoperiod had no effect on milk production after that period. In conclusion, the results do not support to the hypothesis that photoperiod affects mammary gland development during the second allometric phase. However, they confirm that a short-day photoperiod in late gestation enhances milk production in the following lactation in primiparous heifers. Using serum PRL as an indicator of the photoperiodic response, we can conclude that responsiveness to the photoperiodic signal is still conditioned by a previous photoperiod several months after it ends.

Effect of prepartum photoperiod and melatonin feeding on milk production and prolactin concentration in dairy heifers and cows.

Holstein multiparous cows (n = 29) and primiparous heifers (n = 32) calving over a 1-yr period were subjected to photoperiod-melatonin treatments according to a 2 × 3 factorial design. Starting 8 wk before expected calving, all animals were subjected to 1 of the following treatments: 8h of light and 16 h of dark (8L:16D), 16 h of light and 8h of dark (16L:8D), or 16L:8D plus melatonin feeding (16L:8D-melatonin). Each day at 1355 h, the animals in the melatonin treatment received orally a gelatin capsule containing 25mg of melatonin. The treatments ended at calving, when the animals were moved to the lactation barn; all animals were then subjected to about 16 h of light per day. At the beginning and end of the treatment period before calving, blood samples were taken from 6 heifers and 6 cows through a jugular cannula for 24h at 30-min intervals to monitor serum melatonin and prolactin concentrations. Milk production in the heifers was not affected by the photoperiod treatments. Early-lactation milk production was higher in the cows exposed to the short-day photoperiod than in those exposed to a long-day photoperiod (16L:8D and 16L:8D-melatonin), with averages of 36.7 ± 0.9, 33.1 ± 0.8, and 34.1 ± 0.9 kg/d for 8L:16D, 16L:8D, and 16L:8D-melatonin, respectively. Photoperiod had no effect on late-lactation milk production in the cows. During lactation, the dry matter intake of heifers was not affected by the treatments, but dry matter intake of the cows exposed to a short-day photoperiod was greater than that of the cows exposed to a long-day photoperiod. Feed efficiency of heifers was improved by short-day photoperiod. During the treatment period, prolactin concentration was lower in the animals exposed to a short-day photoperiod than in those exposed to a long-day photoperiod, was lower with the 16L:8D-melatonin treatment than with the 16L:8D treatment, and tended to be lower with the 8L:16D treatment than with the 16L:8D-melatonin treatment, with averages of 3.5 ± 0.8, 9.9 ± 0.8, and 6.0 ± 0.8 ng/mL for 8L:16D, 16L:8D, and 16L:8D-melatonin, respectively. In early lactation, prolactin concentration was lower in the heifers exposed to the 16L:8D photoperiod during the dry period than in those exposed to the 8L:16D photoperiod or fed melatonin. In conclusion, a short-day photoperiod during the dry period transiently increases milk production of cows and the feed efficiency of heifers in the following lactation. However, melatonin cannot be used to mimic a short-day photoperiod during the dry period.

Utilization of lactoferrin to fight antibiotic-resistant mammary gland pathogens.

The widespread use of antibiotics has lead to the increased presence of pathogens that are less susceptible to their antibacterial effect. Lactoferrin (Lf) is naturally produced by the mammary gland. Lactoferrin is the main whey protein in human milk and is also present in cow's milk but at a much lower concentration than in human milk. This protein appears to have many biological functions, including antibacterial and antiinflammatory activities. The best-known effect of Lf is to bind iron that is essential for bacterial growth. However, the cationic nature of this protein also appears to be important for the antimicrobial activity of this protein. Lactoferrin has a weak antibacterial effect when used alone, but interestingly, Lf appears much more effective when used at low concentration in combination with several antibiotics. The most striking observation is that Lf increases the inhibitory activity of penicillin up to 4-fold in most penicillin-susceptible Staphylococcus aureus strains, whereas this increase was 4- to 16-fold in penicillin-resistant strains. Indeed, Lf reduces beta-lactamase activity in S. aureus strains producing this enzyme. Transcription of beta-lactamase gene is dramatically repressed in the presence of Lf. We evaluated the efficacy of intramammary treatments containing penicillin G or bovine Lf (bLf), or both, to cure chronic mastitis caused by a clinical isolate of S. aureus highly resistant to beta-lactam antibiotics. In a first trial, mastitis was induced in lactating cows by injecting a low dose of S. aureus through the teat canal of all quarters. Bacterial cure rate was null for control quarters, 11.1% for bLf, 9.1% for penicillin, and 45.5% for the combination of bLf and penicillin. A second trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. Quarters were treated with 100,000 IU of penicillin G with or without 250 mg of bLf for 7 d. Bacterial cure rate was greater for the bLf + penicillin combination (33.3%) compared with penicillin alone (12.5%). In conclusion, bLf added to penicillin is an effective combination for the treatment of stable S. aureus infections resistant to beta-lactam antibiotics.

Efficacy of a lactoferrin-penicillin combination to treat {beta}-lactam-resistant Staphylococcus aureus mastitis.

The efficacy of intramammary (IM) treatments containing penicillin G (PG) alone or a combination of PG and bovine lactoferrin (bLF) was evaluated using a model of experimentally induced chronic bovine mastitis caused by a clinical isolate of Staphylococcus aureus highly resistant to beta-lactam antibiotics. First, we confirmed that this strain could cause mastitis and infection could not be cured with PG alone. In a second trial, chronic mastitis was induced in 19 late-lactating cows by injecting a low dose of Staph. aureus through the teat canal of all quarters. After 15 d, cows with stable infections in their 4 quarters had their mammary quarters randomly assigned, within cow, to 1 of 4 IM treatments as follows: 1) citrate buffer, 2) 100,000 IU of PG, (3) 1 g of bLF, or 4) 1 g of bLF + 100,000 IU of PG. Treatments were repeated twice a day for 5 d. A third trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. One month before dry-off, 20 gravid cows regrouped by dates of calving were infected in their 4 quarters. Once infections were established, cows were dried off abruptly. After calving, aseptic milk samples were collected separately from all quarters for 4 wk to monitor infection. Mammary quarters from enrolled cows were then randomly assigned, within cow, to 1 of 2 treatments as follows: 1) 100,000 IU of PG or 2) 250 mg of bLF + 100,000 IU of PG. Treatments were administered IM twice a day for 7 d. For all trials, milk samples were taken to monitor bacterial concentration and somatic cell count. Bacteriological cure rate was determined using milk samples taken 3 and 4 wk after initiation of treatments. For the second trial, cure rate was null for control quarters, 11.1% for bLF, 9.1% for PG, and 45.5% for the bLF + PG combination. For cows infected in their previous lactation, cure rate was higher for the bLF + PG combination (33.3%) compared with PG alone (12.5%). In conclusion, bLF added to PG is an effective combination (i.e., 3- to 5-times higher cure rate) for the treatment of stable Staph. aureu infections highly resistant to beta-lactam antibiotics.

Effect of stage of lactation and parity on mammary gland cell renewal.

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8 +/- 1.3 and 25.2 +/- 0.8 kg/d) and 50 DIM (38.0 +/- 1.2 and 29.8 +/- 1.1 kg/d), but it was the same for both groups at 250 DIM (23.9 +/- 1.5 and 23.8 +/- 1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, beta-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.

Blood melatonin and prolactin concentrations in dairy cows exposed to 60 Hz electric and magnetic fields during 8 h photoperiods.

Two experiments were conducted to test the hypothesis that electric and magnetic field (EMF) exposure may result in endocrine responses similar to those observed in animals exposed to long days. In the first experiment, 16 lactating, pregnant Holstein cows were assigned to two replicates according to a crossover design with treatment switchback. All animals were confined to wooden metabolic cages and maintained under short day photoperiods (8 h light/16 h dark). Treated animals were exposed to a vertical electric field of 10 kV/m and a horizontal magnetic field of 30 microT (EMF) for 16 h/day for 4 weeks. In a second, similar experiment, 16 nonlactating, nonpregnant Holstein cows subjected to short days were exposed to EMF, using a similar protocol, for periods corresponding to the duration of one estrous cycle. In the first experiment, circulating MLT concentrations during the light period showed a small numerical decrease during EMF exposure (P < .05). Least-square means for the 8 h light period were 9.9 versus 12.4 pg/ml, SE = 1.3. Melatonin concentrations during the dark period were not affected by the treatment. A similar trend was observed in the second experiment, where MLT concentrations during the light period tended to be lower (8.8 pg/ml vs. 16.3 pg/ml, P < .06) in the EMF exposed group, and no effects were observed during the dark period. Plasma prolactin (PRL) was increased in the EMF exposed group (16.6 vs. 12.7 ng/ml, P < .02) in the first experiment. In the second experiment, the overall PRL concentrations found were lower, and the mean plasma PRL concentration was not affected by treatment. These experiments provide evidence that EMF exposure may modify the response of dairy cows to photoperiod.

Impacts of dietary protein level and feed restriction during prepuberty on mammogenesis in gilts.

The possible roles of dietary protein level and feed restriction in regulating mammary development of prepubertal gilts were investigated. Cross-bred gilts were fed a commercial diet until 90 d of age and then divided into four nutritional regimens based on two pelleted diets (as-fed basis): a high-protein diet (HP = 13.8 MJ of ME, 1.0% total lysine, 18.7% CP) and a low-protein diet (LP = 13.8 MJ of ME, 0.7% total lysine, 14.4% CP). Nutritional regimens were as follows: 1) HP ad libitum until slaughter (n = 22, T1); 2) HP ad libitum until 150 d of age followed by LP until slaughter (n = 20, T2); 3) LP ad libitum until slaughter (n = 21, T3); and 4) HP with a 20% feed restriction until slaughter (n = 19, T4). Gilts were weighed, their backfat thickness was measured, and jugular blood samples were obtained on d 90, 150, and at slaughter to determine concentrations of prolactin, IGF-I, leptin, and glucose. Gilts were slaughtered 8+/-1 d after their first or second estrus (202.7+/-14.5 d of age). Mammary glands were excised, parenchymal and extraparenchymal tissues were dissected, and composition of parenchymal tissue (protein, fat, DM, DNA, protein/DNA) was determined. The T4 gilts weighed less (P < 0.01) and had less backfat (P < 0.01) than did gilts on other treatments on d 150 and at slaughter. Treatments had no significant effects on prolactin, IGF-I, or glucose concentrations, but there was a treatment x day interaction (P < 0.01) for leptin, with concentrations being lower at slaughter in restricted-fed (T4) vs. LP (T3) gilts (P < 0.05). There was less extraparenchymal mammary tissue (P < 0.01) in T4 gilts than in gilts from the other groups and a tendency (P = 0.13) for the amount of parenchymal tissue to be lower in T4 gilts. In conclusion, a lower lysine intake during prepuberty did not hinder mammary development of gilts, but a 20% feed restriction decreased mass of parenchymal and extraparenchymal tissues. The effect of feed restriction on extraparenchymal tissue is most likely associated with the lower fat deposition.

Lack of effect of 10 kV/m 60 Hz electric field exposure on pregnant dairy heifer hormones.

Sixteen pregnant Holstein heifers weighing 521 +/- 46 kg, at 3.3 +/- 0.7 months of gestation and 2.2 +/- 2.0 months of age were confined to wooden metabolism cages and were exposed to a vertical electric field (EF) of 10.0 +/- 0.4 kV/m and an artificial light cycle of 12 h light-12 h dark. The heifers were divided into two replicates of eight each. Each replicate was divided into two groups of four animals each, one group becoming the non-exposed and the second, the EF exposed group. The exposed group were housed in metabolism cages in an area where EF were generated, and the non-exposed group, in metabolism cages located in the adjacent area where the EF was less than 2% of that present in the exposed area. The test animals were subject to the different treatments for 4 weeks continuously. After 4 weeks, the animals switched treatment, the exposed group becoming the non-exposed group and vice-versa. Then the treatment continued for 4 more weeks. Catheters were inserted into the jugular vein of the animals, and blood samples were collected on twice a week to estimate the serum concentration of progesterone (P4), melatonin (MLT), prolactin (PRL), and insulin-like growth factor-1 (IGF-1). Feed consumption was measured daily and feed samples were collected twice a week. The results indicated that exposure of dairy cattle to EF similar to those encountered directly underneath a 735 kV high tension electrical power line carrying a maximum load of current, cannot be associated with any variation in the experimental variables mentioned above. An exception to this, is the variation in MLT, which was associated with the EF exposure. Due to the inconsistency of the MLT response in the different replicates, caution should be exercised in the interpretation of this phenomenon.

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.

Specific window of prolactin inhibition in late gestation decreases mammary parenchymal tissue development in gilts.

Prolactin is required from d 70 to 110 of gestation for normal mammary development of gilts. The goal of the present study was to determine the effect of inhibiting prolactin with bromocriptine during specific time windows during the second half of gestation on mammary gland development in gilts. Crossbred primigravid gilts were assigned as controls (n = 12) or received 10 mg of bromocriptine orally three times daily from d 50 to 69 (BR50, n = 12), d 70 to 89 (BR70, n = 12), or d 90 to 109 (BR90, n = 12) of gestation. Jugular blood samples were collected on d 50, 70, 90, and 109 of gestation and assayed for prolactin and estradiol. Gilts were slaughtered on d 109 of gestation and fetuses were counted and weighed. One row of mammary glands was used for dissection of parenchymal and extraparenchymal tissues, and for biochemical analyses. Tissue from the other row was used for measures of prolactin receptor number and affinity. Concentrations of prolactin were decreased markedly (P < 0.001) at the end of each bromocriptine treatment period compared with controls, but there was no overall treatment effect (P > 0.1) on estradiol concentrations. Extraparenchymal tissue weight of the mammary glands was unaffected by treatments (P > 0.1), but weight of parenchymal tissue, total DNA, and total RNA were lower (P < 0.01) in BR90 than control gilts. The percentage of DM in parenchymal tissue was unaffected by treatments (P > 0.1), but percentage of fat was higher and percentage of protein lower (P < 0.01) in BR90 gilts compared with controls. Cell size, as estimated by the protein:DNA ratio, also was lower (P < 0.01) in the BR90 group. Number and affinity of prolactin receptors in parenchymal tissue were not significantly altered by treatments. In conclusion, there is a specific time period in the second half of gestation, from 90 to 109 d, during which prolactin is essential for normal mammary parenchymal tissue development.

Responses of the estrous cycle in dairy cows exposed to electric and magnetic fields (60 Hz) during 8-h photoperiods.

To study the effects of exposure to extremely low frequency (ELF) electric and magnetic fields (EMF) on the estrous cycle of dairy cows under short-day photoperiod, 16 non-lactating, non-pregnant Holstein cows were exposed to a vertical electric field of 10 kV/m and a horizontal magnetic field of 30 microT for 16 h per day in a cross-over design consisting of two sequences. Each sequence included three periods, and each period corresponded to the duration of one estrous cycle. All animals were maintained under short photoperiod (8 h light/16 h dark) during the trial. Exposure to EMF had an impact on the duration of a complete estrous cycle (P<0.01) and on the duration of the luteal phase (P<0.01). The mean duration of one cycle was 19.5+/-0.4 for the control and 21.3+/-0.4 days for the exposed animals, respectively. The mean duration of the luteal phase was 15.4+/-0.4 days for the control and 17.2+/-0.4 days for the exposed group. The total area under the progesterone (P(4)) curve, the amplitude of the curve or the slope of the P(4) rise at the onset of the luteal phase were not affected by EMF exposure. Results indicate that exposure to EMF may increase the duration of the estrous cycle.

Management of photoperiod in the dairy herd for improved production and health.

Environmental influences on lactation efficiency are frequently associated with reductions in milk output. Heat stress, for example, leads to depressed feed intake and, subsequently, losses in production. Conversely, cold stress may limit nutrients available for milk synthesis. Fortunately, one environmental factor, photoperiod, can exert a positive effect on dairy performance when managed properly. Long days have consistently been shown to improve milk yield during established lactation. In addition, photoperiod management can be used to improve heifer growth and maximize accretion of lean tissue, including mammary parenchyma. There is, however, evidence of refractoriness to long day stimulation. Recent work has focused on the dry period as a time when photoperiod manipulation can influence subsequent milk production. In contrast to lactating cows, multiparous cows benefit from exposure to short days when the dry period is followed by long days or natural photoperiod after calving. Similarly, primiparous animals also respond positively to short days late in pregnancy when subsequently exposed to long days during lactation. Furthermore, emerging evidence suggests that short days positively influence immune function in cattle. Mechanistically, it appears that prolactin has a causal relationship with the observed dairy performance effects during the dry period and on immune function, via altered sensitivity to prolactin through differential expression of prolactin receptor in multiple tissues. The objectives of this paper include a review of fundamental aspects of photoperiod physiology, integration of applied and basic research findings, and development of management recommendations for the entire life cycle of the dairy cow to optimize performance.

Effect of electric and magnetic fields (60 Hz) on production, and levels of growth hormone and insulin-like growth factor 1, in lactating, pregnant cows subjected to short days.

Electric and magnetic fields (EMF) are generated by the transmission of electricity through high tension lines traversing rural areas. Previous studies showed increased dry matter intake (DMI) and fat corrected milk in dairy cows exposed to EMF. Because EMF exposure has been shown to suppress pineal release of melatonin in some species, it was hypothesized that EMF effects resemble those of exposure to long days. Previous studies have shown that DMI and milk production increase in dairy cattle in response to long day photoperiods, and this has been observed in association with increased circulating insulin-like growth factor 1 (IGF-1), but not growth hormone (GH). The hypothesis that EMF act by modifying the response to photoperiod was tested by subjecting dairy cows to controlled EMF exposure while keeping them under short-day conditions. Sixteen lactating, pregnant Holstein cows were exposed to a vertical electric field of 10 kV/m and a horizontal magnetic field of 30 microT in a crossover design with treatment switchback. Two groups of eight cows each were exposed to EMF for 16 h/d in either oftwo sequences. Each sequence consisted of three consecutive 28-d periods. All animals were maintained under short day conditions (8 h light, 16 h dark) during the trial. DMI and plasma IGF-1 were increased (P < 0.01) during EMF exposure (17.03 vs.16.04 kg/d, SE = 0.4; 137 +/- 6 ng/ml vs 126 +/- 6, respectively). The mean GH concentration was not affected, but a treatment x hour interaction was detected, with GH lower for the EMF exposed animals during the first 16 h of the sampling period, and higher for the last 8 h. Overall, the yield of milk or its components was not affected by EMF exposure, but milk yield was significantly higher for the exposed animals during wk 4 of treatment.

Variations in mammary protein metabolism during the natural filling of the udder with milk over a 12-h period between two milkings: leucine kinetics.

To define the temporal variations of whole body and mammary leucine kinetics over a 12-h period between two milkings, we used two groups of four Holstein cows, one in their second and the other in their third or fourth lactation. Cows were infused with L-[1-13C]leucine during the 12-h interval between two milkings. Blood was sampled every 30 min during that period from arterial and mammary sources. Timeafter milking did not affect whole body irreversible loss rate of leucine but affected whole body leucine oxidation, which broadly followed variations in arterial plasma leucine concentration. Similarly, mammary leucine irreversible loss rate and leucine used for protein synthesis were not affected by time after milking. Leucine oxidation by the mammary gland was, however, affected by time after milking. It increased by 15% from the first 2-h period to the following 4-h period and then decreased by 13% over the following 2-h period. A 21% increase in leucine oxidation was observed from 8 to 10 h after milking, and then it decreased by 26% over the last 2-h period. Protein degradation expressed as percentage of mammary leucine flux followed a similar temporal pattern. Leucine used for protein synthesis by the mammary gland was unaltered over time after milking, suggesting that the increased availability of leucine resulting from mammary protein breakdown would increase intracellular concentrations of leucine, which would have favored its catabolism. Overall, these results confirm the high metabolic activity of the mammary gland, as protein synthesis by the mammary gland averaged 43% of whole body protein synthesis.

Response of Staphylococcus aureus isolates from bovine mastitis to exogenous iron sources.

Staphylococcus aureus can survive in conditions of extremely low iron concentration. The ability of S. aureus to use two exogenous hydroxamate types of siderophores (desferrioxamine and ferrichrome) and four iron-containing proteins found in cattle (hemin, hemoglobin, ferritin, and lactoferrin) were tested on 16 reference and clinical isolates. For all strains tested, ferrichrome and desferrioxamine showed strong growth-promoting activities in a disk diffusion assay and in liquid medium. The heme proteins hemin and hemoglobin were also found to support growth in culture media lacking other iron sources, while lactoferrin failed to do so. On media containing the iron chelator dipyridyl, ferritin induced a growth inhibition effect that was further enhanced in the presence of lactoferrin in seven of the 13 tested strains. Staphylococcus aureus was able to bind hemin and the level of binding activity was not increased after growth in iron-rich or -poor media. Dot-blot competition tests showed that biotin-labeled lactoferrin binds to S. aureus, and this binding can be inhibited by unlabeled lactoferrin. Expression of lactoferrin-binding activity was independent of the level of iron in the medium and the iron saturation status of lactoferrin. For each strain tested, ligand blots showed lactoferrin-binding proteins of molecular weights ranging from 32 to 92 kDa. Possible functions of these lactoferrin-binding proteins could not be related to iron acquisition mechanism in S. aureus.

Variations in mammary metabolism during the natural filling of the udder with milk over a 12-h period between two milkings.

Two groups of four Holstein cows, one in their second and the other in their third or fourth lactation, were used to study temporal variations of mammary metabolism over a 12-h period between two milkings. Blood samples were collected every 30 min from an artery and a mammary vein during a 12-h interval between two milkings. Isoleucine, leucine, lysine, methionine, and phenylalanine mammary net fluxes varied or tended to change over time after milking with a similar pattern between whole blood and plasma. For these amino acids, whole blood and plasma net fluxes reached their maximum over the first 8 h after milking. Simultaneously, respiratory quotients decreased linearly and varied from 2.31 to 2.01 during the first 8 h of the period, suggesting active mammary lipogenesis. From 8 to 12 h after milking, mammary amino acid net fluxes decreased, while mammary oxygen uptake tended to increase with a concomitant decrease in the respiratory quotient reaching 1.84 to 1.40. These findings suggest that, beginning 8 h after milking, mammary uptake of amino acids starts to decrease and catabolic processes appear promoted; this phenomenon could help to explain the increase in milk production reported in the literature with increased milking frequency.

Delineating potential control mechanisms of mammary protein synthesis utilizing atropine.

Amino acids (AA) and/or somatotropin (ST) were infused into a jugular vein of lactating cows receiving atropine to determine the effect on milk protein secretion. Atropine decreased milk protein yield by 35%. Plasma alpha-amino nitrogen, which was decreased by 31% due to atropine, was restored to control levels upon AA infusion. Plasma ST concentration was not affected by atropine and, upon ST infusion, increased by 236% in treated animals. Amino acids, ST, or the combination of the two were unable to return milk protein to control levels in atropine-treated animals. Plasma insulin concentration decreased by 37% in atropine-treated animals and was not significantly different for cows receiving atropine as well as ST, AA, or both ST and AA. Similarly, glucagon decreased by 36% in atropine-treated animals but was partially restored in cows receiving AA. Overall, the insulin-to-glucagon ratio was not significantly affected by treatment. Plasma glucose concentration was not affected by treatment. These data lend support to the importance of the insulin-to-glucagon ratio in terms of whole-body metabolism, with the exception of the mammary glands, which are glucagon-insensitive and, therefore, sensitive to the observed decrease in circulating insulin concentration.

Effect of lactoferrin in combination with penicillin on the morphology and the physiology of Staphylococcus aureus isolated from bovine mastitis.

The objective of the present study was to evaluate the therapeutic potential of bovine lactoferrin or lactoferricin in combination with penicillin G against Staphylococcus aureus. Minimal inhibitory concentrations of lactoferrin, lactoferricin, penicillin, and combinations of lactoferrin or lactoferricin with penicillin were determined for 15 S. aureus strains including several strains resistant to beta-lactam antibiotics. The fractional inhibitory concentration index indicated a synergistic effect between lactoferrin and penicillin. Combination of lactoferrin with penicillin increased the inhibitory activity of penicillin by two- to fourfold and reduced the growth rate in S. aureus strains tested, whereas the increase in the inhibitory activity of lactoferrin by penicillin was 16- to 64-fold. The addition of iron to the medium containing a combination of penicillin and lactoferrin had no effect on growth inhibition. Electron microscopy revealed that concentration below the minimal inhibitory concentrations of penicillin induced important ultrastructure alterations, which were further enhanced by the presence of lactoferrin. When S. aureus cells were grown in the presence of a combination of penicillin and lactoferrin, changes in the protein profile of the bacteria, including the disappearance of several protein bands due to the presence of lactoferrin, were observed. These data suggest that bovine lactoferrin or lactoferricin in combination with beta-lactam antibiotics can increase the antibacterial activity of these antibiotics against S. aureus resistant to antibiotics.

Effects of insulin, recombinant bovine somatotropin, and their interaction on insulin-like growth factor-I secretion and milk protein production in dairy cows.

This trial was designed to test the effects of insulin, recombinant bovine somatotropin (rbST), and their interaction on milk protein and selected blood parameters in dairy cows. Eight Holstein cows (86 +/- 10 d in milk) were divided in two groups and used in two replicates of a Latin square design with four animals, four periods, and four treatments: 1) intravenous infusion of saline, 2) infusion of saline and subcutaneous administration of 40 mg of rbST per day, 3) intravenous infusion of 12 mg of insulin per day coupled with glucose infusion, and 4) rbST administration combined with insulin and glucose infusion. The glucose infusion rate was adjusted to maintain euglycemia. Each experimental period lasted 14 d: treatments were administered during the first 6 d, and no treatment was administered during the following 8-d resting phase. The average daily amount of glucose infusion needed to avoid hypoglycemia was 2.8 kg/cow when only insulin was infused as opposed to 2.2 kg/cow when both insulin and rbST were administered, indicating that either rbST causes a peripheral resistance to insulin or rbST increased liver gluconeogenesis or both. Data from the last 3 d of infusion were analyzed by using the SAS system for mixed models. Percent protein of milk tended to be lower (2.84 vs. 2.79%) and milk urea content was lower (16.6 vs. 14.8 mg/dl) during rbST administration, regardless of insulin infusion. Insulin infusion increased percent protein (2.78 vs. 2.85%) and percent casein (2.36 vs. 2.46%) and decreased milk urea content (17.1 vs. 14.3 mg/dl) regardless of rbST administration. For milk yield, protein yield, casein yield, lactose percent, and lactose yield, there were significant interactions between insulin and rbST administration. For example, casein yield averaged 1.17, 1.12, 1.20, and 1.28 kg/d for saline, insulin, rbST, and insulin combined with rbST, respectively. Similarly, there was a significant interaction between insulin and rbST on IGF-I levels, which were 122.5, 181.3, 342.3, and 492.2 ng/ml for saline, insulin, rbST, and insulin combined with rbST, respectively. In conclusion, these results clearly demonstrated that insulin interacts with bST in early lactation to improve milk protein synthesis and yield in dairy cows. These effects are probably mediated through a combination of bST nutrient mobilization, bST-induced gluconeogenesis, bST-induced insulin peripheral resistance, and bST/insulin synergism on insulin-like growth factor-I secretion and on mammary epithelial tissue.