Short communication: Bovine α-casein is a ferritin-binding protein and inhibitory factor of milk ferritin immunoassay

Commercial bovine milk α-casein, but not β- and κ-caseins, bound to bovine spleen ferritin, as determined by an immunoassay for ferritin. In contrast, α-casein did not bind to apoferritin. The binding of α-casein to bovine spleen ferritin was strongly inhibited by increasing ionic strength by the addition of 0.5 M (NH₄)₂SO₄. The addition of α-casein to a known amount of bovine spleen ferritin resulted in significantly lower recovery (78-80%) of added ferritin, although β- and κ-caseins showed little inhibitory effect in the ferritin immunoassay. These results indicate that bovine α-casein is a specific ferritin-binding protein that may inhibit milk ferritin immunoassay.     

Concentrations of 17beta-estradiol in Holstein whole milk

Some individuals have expressed concern about estrogens in food because of their potential to promote growth of estrogen-sensitive human cancer cells. Researchers have reported concentrations of estrogen in milk but few whole milk samples have been analyzed. Because estrogen associates with the fat phase of milk, the analysis of whole milk is an important consideration. The objectives of this study, therefore, were to quantify 17β-estradiol (E₂) in whole milk from dairy cows and to determine whether E₂ concentrations in milk from cows in the second half of pregnancy were greater than that in milk from cows in the first half of pregnancy or in nonpregnant cows. Milk samples and weights were collected during a single morning milking from 206 Holstein cows. Triplicate samples were collected and 2 samples were analyzed for fat, protein, lactose, and somatic cell counts (SCC); 1 sample was homogenized and analyzed for E₂. The homogenized whole milk (3 mL) was extracted twice with ethyl acetate and once with methanol. The extract was reconstituted in benzene:methanol (9:1, vol/vol) and run over a Sephadex LH-20 column to separate E₂ from cholesterol and estrone before quantification using radioimmunoassay. Cows were classified as not pregnant (NP, n = 138), early pregnant (EP, 1 to 140 d pregnant, n = 47), or midpregnant (MP, 141 to 210 d pregnant, n = 21) at the time of milk sampling based on herd health records. Mean E₂ concentration in whole milk was 1.4 ± 0.2 pg/mL and ranged from nondetectable to 22.9 pg/mL. Milk E₂ concentrations averaged 1.3, 0.9, and 3.0 pg/mL for NP, EP, and MP cows, respectively. Milk E₂ concentrations for MP cows were greater and differed from those of NP and EP cows. Milk composition was normal for a Holstein herd in that log SCC values and percentages of fat, protein, and lactose averaged 4.9, 3.5, 3.1, and 4.8, respectively. Estradiol concentration was significantly correlated (r = 0.20) with percentage fat in milk. Mean milk yield was 18.9 ± 0.6 kg for the morning milking. The mean E₂ mass accumulated in the morning milk was 23.2 ± 3.4 ng/cow. Likewise, using the overall mean concentration for E₂ in milk, the mean E₂ mass in 237 mL (8 fluid ounces) of raw whole milk was 330 pg. The quantity of E₂ in whole milk, therefore, is low and is unlikely to pose a health risk for humans.     

Short communication: Effect of estrogen supplemented at dry-off on temporal changes in concentrations of lactose in blood plasma of Holstein cows

The objective was to determine the effect of supplemental estrogen (estradiol cypionate, ECP) at dry-off on temporal changes in concentrations of lactose in blood plasma of Holstein cows as an indicator of rate of mammary involution. Thirty-two Holstein cows (8/group) were assigned randomly to 4 treatment groups: 30-d dry, 30-d dry + ECP, 60-d dry, and 60-d dry + ECP. A single injection (7.5 mL) of cottonseed oil (30- and 60-d dry) or ECP (15 mg) in oil (30- and 60-d dry + ECP) was administered intramuscularly at dry-off. Blood samples were collected from the coccygeal vein of all cows 24 h before dry-off and at dry-off, and then 8 samples were collected throughout the subsequent 48 h to monitor concentrations of lactose in blood plasma. No significant effects of ECP on the overall mean concentrations of lactose were detected. Concentrations of lactose increased and were greatest in blood collected 20 h (520.4 ± 54.1, 268.1 ± 48.2, 345.0 ± 52.3, 418.4 ± 49.8 μM, for the 4 treatment groups respective to the order listed above) after supplemental ECP and final milk removal. At 40 h, concentrations approached those observed 24 h before dry-off (140.5 ± 52.1, 57.6 ± 47.1, 90.1 ± 51.4, 61.2 ± 48.4 μM, respectively). Concentrations of lactose at 20 h were positively correlated with milk yield of cows at dry-off. Similar temporal profiles of lactose in blood plasma of cows supplemented or not with ECP indicated that ECP at dry-off did not markedly alter the course of tight junction leakage that typically occurs in mammary epithelial tissue during progressive early involution when milk removal is discontinued.     

Characterization of polyphenol oxidase from broccoli (Brassica oleracea var. botrytis italica) florets

Polyphenol oxidase (PPO) from broccoli florets was extracted and purified through (NH₄)₂SO₄ precipitation, ion-exchange and gel filtration chromatography. The molecular weight was estimated to lie between 51.3 and 57 kDa by sodium dodecyl sulphate-polyacrylamide gel electophoresis (SDS-PAGE) and gel filtration. The effects of substrate specificity, pH, and sensitivity to various inhibitors: citric acid, ascorbic acid, sodium sulphate and EDTA (sodium salt of ethylenediaminetetraacetic acid) of partially purified PPO were investigated. Polyphenol oxidase showed the best activity toward catechol (K_M = 12.34 ± 0.057 mM, V_max = 2000 ± 8736 U/ml/min) and 4-methyl catechol (K_M = 21 ± 0.087 mM, V_max = 28.20 ± 0.525 U/ml/min). The optimum pH for broccoli PPO was 5.7 with catechol and 4-methylcatechol as substrates. The most effective inhibitor was sodium sulphate.     

Effect of forage-to-concentrate ratio in dairy cow diets on emission of methane, carbon dioxide, and ammonia, lactation performance, and manure excretion

Holstein cows housed in a modified tie-stall barn were used to determine the effect of feeding diets with different forage-to-concentrate ratios (F:C) on performance and emission of CH₄, CO₂ and manure NH₃-N. Eight multiparous cows (means ± standard deviation): 620 ± 68 kg of body weight; 52 ± 34 d in milk and 8 primiparous cows (546 ± 38 kg of body weight; 93 ± 39 d in milk) were randomly assigned to 1 of 4 air-flow controlled chambers, constructed to fit 4 cows each. Chambers were assigned to dietary treatment sequences in a single 4 × 4 Latin square design. Dietary treatments, fed as 16.2% crude protein total mixed rations included the following F:C ratio: 47:53, 54:46, 61:39, and 68:32 [diet dry matter (DM) basis]. Forage consisted of alfalfa silage and corn silage in a 1:1 ratio. Cow performance and emission data were measured on the last 7 d and the last 4 d, respectively of each 21-d period. Air samples entering and exiting each chamber were analyzed with a photo-acoustic field gas monitor. In a companion study, fermentation pattern was studied in 8 rumen-cannulated cows. Increasing F:C ratio in the diet had no effect on DM intake (21.1 ± 1.5 kg/d), energy-corrected milk (ECM, 37.4 ± 2.2 kg/d), ECM/DM intake (1.81 ± 0.18), yield of milk fat, and manure excretion and composition; however, it increased milk fat content linearly by 7% and decreased linearly true protein, lactose, and solids-not-fat content (by 4, 1, and 2%, respectively) and yield (by 10, 6, and 6%, respectively), and milk N-to-N intake ratio. On average 93% of the N consumed by the cows in the chambers was accounted for as milk N, manure N, or emitted NH₃-N. Increasing the F:C ratio also increased ruminal pH linearly and affected concentrations of butyrate and isovalerate quadratically. Increasing the F:C ratio from 47:53 to 68:32 increased CH₄ emission from 538 to 648 g/cow per day, but had no effect on manure NH₃-N emission (14.1 ± 3.9 g/cow per day) and CO₂ emission (18,325 ± 2,241 g/cow per day). In this trial, CH₄ emission remained constant per unit of neutral detergent fiber intake (1 g of CH₄ was emitted for every 10.3 g of neutral detergent fiber consumed by the cow), but increased from 14.4 to 18.0 g/kg of ECM when the percentage of forage in the diet increased from 47 to 68%. Although the pattern of emission within a day was distinct for each gas, emissions were higher between morning feeding (0930 h) and afternoon milking (1600 h) than later in the day. Altering the level of forage within a practical range and rebalancing dietary crude protein with common feeds of the Midwest of the United States had no effects on manure NH₃-N emission but altered CH₄ emission.     

Supplementation of progesterone via controlled internal drug release inserts during ovulation synchronization protocols in lactating dairy cows

Our objective was to determine the effect of exogenous progesterone (P4) during a timed artificial insemination (TAI) protocol on pregnancies per AI (P/AI) in dairy cows not previously detected in estrus. Lactating cows (n = 3,248) from 7 commercial dairy herds were submitted to a presynchronization protocol (2 injections of PGF_2α 14 d apart; Presynch), and cows in estrus after the second PGF_2α received AI (EDAI; n = 1,583). Cows not inseminated by 12 to 14 d after the second PGF_2α injection were submitted to a TAI protocol (GnRH on d 0, PGF_2α on d 7, and GnRH + TAI 72 h after PGF_2α). At onset of the TAI protocol, cows were balanced by parity and days in milk and assigned randomly to receive no exogenous P4 (control, n = 803) or a controlled internal drug release (CIDR) insert containing 1.38 g of P4 from d 0 to 7 (CIDR, n = 862). Blood samples were collected at the second PGF_2α injection of the Presynch and on the day of the first GnRH injection of the TAI protocol for P4 determination. When P4 in both samples was <1 ng/mL, cows were classified as anovular, whereas cows having at least 1 sample ≥1 ng/mL were classified as cyclic. Concentration of P4 at 11 to 14 d after AI was determined in a subgroup of cows (n = 453) from 2 herds. Pregnancy was diagnosed at 40 ± 5 and 65 ± 5 d after AI. Proportion of cows inseminated on estrus after the second PGF_2α injection of the Presynch protocol differed among herds (range = 26.7 to 59.8%). Overall P/AI for EDAI cows at 40 ± 5 and 65 ± 5 d were 36.2 and 33.7%, respectively, and pregnancy loss was 8.8%. Proportion of cyclic cows at the onset of the TAI protocol differed among herds (range from 66.5 to 86.3%), but did not differ between treatments (control = 72.4%, CIDR = 74.1%). Treatment affected P/AI at 40 ± 5 (control = 33.3%, CIDR = 38.1%) and 65 ± 5 (control = 30.0%, CIDR = 35.1%) d after AI but did not affect pregnancy loss (8.6%). Cyclic cows had greater P/AI at 40 ± 5 (38.2 vs. 29.3%) and 65 ± 5 d (35.1 vs. 26.1%) after AI, but cyclic status had no effect on pregnancy loss. Treatment affected P4 concentration after AI, with more CIDR cows having P4 ≥1 ng/mL (94.4 vs. 86.9%) and P4 ≥3.2 ng/mL (81.8 vs. 68.0%) at 11 to 14 d after AI compared with control cows. Treatment of cows not previously detected in estrus with a CIDR insert during a TAI protocol increased proportion of cows with functional CL after AI and P/AI.     

Milking frequency, estradiol cypionate, and somatotropin influence lactation and reproduction in dairy cows

Our objectives were to determine lactational and reproductive outcomes in response to increased milking frequency (MF), injection of estradiol cypionate (ECP), and treatment with bovine somatotropin (bST). Lactating dairy cows (n = 144) were blocked by lactation number (1 vs. 2+) and assigned randomly to a 2 × 2 × 2 factorial experiment consisting of 8 treatment combinations: 1) MF consisting of 4× daily milking (4×) for the first 30 d in milk (DIM) vs. 2× daily milking (2×), with all cows milked 2× after 30 DIM; 2) 10 mg of ECP given postpartum at 8 ± 3 DIM versus controls that received ECP diluent (oil); and 3) biweekly bovine somatotropin (bST), starting sometime after 60 DIM, versus no bST. Ovulation before the first artificial insemination was synchronized by using Heatsynch (GnRH injection 7 d before PGF_2α followed in 24 h by ECP), and cows were artificially inseminated after detected estrus or at 48 h after ECP, whichever came first. Pregnancy was assessed by transrectal ultrasonography 28 to 30 d after artificial insemination. Daily yield and weekly components of milk were measured during the first 90 DIM. Intervals to first and second postpartum ovulation were unaffected by treatment, but cows were in estrus earlier after 2× (24 ± 4 d) than 4× (41 ± 4 d) daily MF, and sooner after ECP (25 ± 3 d) than after oil (39 ± 4 d) treatment. Pregnancy rates among 4× cows increased for ECP versus oil (52.8 vs. 27.8%) more than for cows with 2× MF treated with ECP versus oil (50.0 vs. 39.4%). Increased MF increased daily milk yields and energy-corrected milk yields during the first 30 DIM. Although milk yields were increased acutely by ECP during the 10 d after its injection, subsequent milk yields were decreased for ECP-treated cows previously milked 4× daily. Treatment with bST increased overall daily milk yields most in cows previously milked 2× daily and treated with oil and those milked 4× daily and treated with ECP. We concluded that early postpartum ECP injection increased pregnancy rates, but generally had detrimental effects on milk yields after 30 DIM for ECP-treated cows previously milked 4× daily, unless those cows also were treated with bST.     

Lactoperoxidase activity in milk is correlated with somatic cell count in dairy cows

Lactoperoxidase (LPO) is a milk protein with antimicrobial function. The present study was undertaken to examine the correlation between LPO activity and somatic cell count (SCC) in milk to use LPO activity as an indicator of mastitis. Composite milk of 36 cows and quarter milk of 3 cows were collected once per week from 0 to 300 d postpartum and twice per day for 1 wk, respectively. For the measurement of LPO activity, milk was mixed with tetramethylbenzidine solution and incubated at 37°C for 30 min, followed by the measurement of optical density. When only milk with low SCC (132 ± 12 × 10³ cells/mL) was used, a significant decrease in LPO activity was detected in primiparous cows from 0 to 4 mo postpartum. Lactoperoxidase activities of primiparous cows in mo 1, 2, and 3 postpartum were significantly higher than those in multiparous cows. When composite milk was divided based on LPO activity, the SCC was significantly higher in the groups with LPO activity >5 and from 3 to 3.9 U/mL in the second- and fourth-parity cows, respectively, compared with the group with LPO activity <2 U/mL. Extremely high SCC were found in the ≥fifth-parity cows, even in low-LPO activity groups. In the case of quarter milk, higher LPO activity was associated with increased SCC in all 3 cows. The percentage of quarter milk samples with high SCC (4,062 ± 415 × 10³ cells/mL) increased with an increase in the LPO activity. The percentage of quarter milk samples with high SCC was 50.0 to 100% in the milk with LPO activity ≥5 U/mL. These results indicate that the correlation of LPO activity to the SCC in bovine milk may point to the potential use of the former as an indicator of SCC.     

Prevotella bryantii 25A used as a probiotic in early-lactation dairy cows: effect on ruminal fermentation characteristics, milk production, and milk composition

Ingestion of high levels of rapidly fermented carbohydrates after parturition often leads to the production of excessive quantities of organic acids that may exceed the buffering capacity of the rumen and cause pH to drop. Ruminal acidosis results in animal discomfort, anorexia, depression, decreased digestibility, and decreased milk production. In the present study, we examined the effects of daily addition of cells of a newly isolated strain of Prevotella bryantii (25A) to the rumen of 12 ruminally cannulated cows in early lactation. This strain was selected based on earlier in vitro studies that indicated its ability to grow rapidly, compete for starch, and produce organic acids other than lactate. After calving, all cows received increasing amounts of an energy-dense diet containing barley grain, corn silage, and grass silage in a 40:60 forage-to-concentrate ratio. Animals were blocked according to milk production from their previous lactation. Treatments (control and P. bryantii) were distributed among cows within the same block. Cows were fed once a day. Six cows were given a daily dose of P. bryantii (2 × 10¹¹ cells/dose), administered directly with a syringe through the rumen cannula, from 3 wk prepartum up to 7 wk postpartum. Rumen fluid was sampled before feeding and at 2 and 3 h postfeeding on wk 1, 2, 3, 4, 6, and 7 postpartum. Feed intake and milk yield were recorded daily and milk composition was recorded 2 d/wk, up to wk 7 of lactation. Feed intake was similar between control and treated cows. Prevotella bryantii did not change milk production, but milk fat tended to be greater in treated cows compared with control cows (3.9 vs. 3.5%). Rumen pH was similar between the 2 groups and differed across sampling times, being higher before feeding (6.3) as opposed to 2 h (5.9) and 3 h (5.7) postfeeding. Rumen lactate concentration was similar before feeding between control and treated cows; however, 2 to 3 h after feeding, lactate concentration was lower in cows receiving P. bryantii compared with control cows (0.7 vs. 1.4 mM). This difference was maintained throughout the experimental period. Concentration of NH₃-N was greater in treated cows than in control cows (174 vs. 142 mg/L). Acetate (65.5 vs. 57.8 mM), butyrate (12.7 vs. 10.5 mM), and branched-chain C4 fatty acid (0.90 vs. 0.75 mM) concentrations were greater in postfeeding samples of treated cows compared with control cows. Supplementing early-lactating cows with P. bryantii 25A increased ruminal fermentation products and milk fat concentration. Because signs of subacute ruminal acidosis were not observed in either treated or control cows, no conclusions can be made about possible protection against acidosis by P. bryantii.     

Effect of supplementing myristic acid in dairy cow rations on ruminal methanogenesis and fatty acid profile in milk

The objective of this study was to evaluate the effects of supplementing myristic acid in dairy cow rations on ruminal methanogenesis and the fatty acid profile in milk. Twelve multiparous Holstein dairy cows (710 ± 17.3 kg of live weight; 290 ± 41.9 d in milk) housed in a tie-stall facility were used in the study. The cows were paired by parity and days in milk and allocated to 1 of 2 treatments: 1) the regular milking cow total mixed ration (control diet), and 2) the regular milking cow total mixed ration supplemented with 5% myristic acid on a dry matter basis (MA diet). The cows were fed and milked twice daily (feeding, 0830 and 1300 h; milking, 0500 and 1500 h). The experiment was conducted as a completely randomized design and consisted of a 7-d pretrial period when cows were fed the control diet to obtain baseline measurements, a 10-d dietary adaptation period, and a 1-d, 8-h measurement period. The MA diet reduced methane (CH₄) production by 36% (608.2 vs. 390.6 ± 56.46 L/d, control vs. MA diet, respectively) and milk fat percentage by 2.4% (4.2 vs. 4.1 ± 0.006%, control vs. MA diet, respectively). The MA diet increased 14:0 in milk by 139% and cis-9 14:1 by 195%. There was a correlation (r = −0.58) between the 14:0 content in milk and CH₄ production and cis-9 14:1 and CH₄ production (r = −0.47). Myristic acid had no effect on the contents of CLA or trans-10 18:1 and trans-11 18:1 isomers in milk. These results suggest that MA could be used to inhibit the activities of methanogens in ruminant animals without altering the conjugated linoleic acid and trans-18:1 fatty acid profile in milk.     

Luteolytic effects of cloprostenol sodium in lactating dairy cows treated with G6G/Ovsynch

The probability of a pregnancy decreases substantially in lactating dairy cows treated with Ovsynch if luteolysis is delayed or incomplete. Two PGF_2α products are currently approved in the United States for luteolysis in lactating dairy cattle, dinoprost tromethamine and cloprostenol sodium. Cloprostenol has a longer half-life compared with dinoprost, is more resistant to endogenous metabolism, and is maintained in circulation longer. We hypothesized that cloprostenol could reduce the time to complete luteolysis compared with dinoprost because of differences in half-life. Lactating dairy cows received the same presynchronization strategy (G6G; 25 mg of PGF_2α - 2 d - 100 μg of GnRH - 6 d - 100 μg of GnRH - 7 d - final PGF_2α treatment). At the time of the final PGF_2α, cows (n = 35) were randomly assigned to receive either 500 μg of cloprostenol or 25 mg of dinoprost. Blood samples were collected daily before and serially after PGF_2α treatment to analyze circulating concentrations of progesterone (P₄) and estradiol (E₂). Ultrasound examinations of ovaries were performed to measure sizes of follicles and corpora lutea (CL) and determine time of ovulation. Considering only cows with complete luteolysis, mean circulating P₄ was lower for cows given cloprostenol than for those given dinoprost between 0 and 12 h postinjection, but not at 24, 36, or 48 h. A rapid decrease in P₄ was observed 1 h after PGF_2α (6.54 ± 0.27 to 3.77 ± 0.22 ng/mL) followed by a complete rebound 1 h later (3.77 ± 0.22 to 5.07 ± 0.31 ng/mL) followed by a steady decline in both treatment groups. Serum concentrations of E₂ were greater at 48 h posttreatment in cloprostenol-treated cows (2.74 ± 0.15 pg/mL) than in dinoprost-treated cows (2.37 ± 0.19 pg/mL). Cows that did not have complete luteolysis did not ovulate (0/7) during the 6-d period following treatment. Time to complete luteolysis and ovulation was 29.1 ± 1.1 versus 29.4 ± 1.7 and 101 versus 103 h posttreatment in cloprostenol compared with dinoprost. A negative relationship was observed between P₄ at 12 h posttreatment and concentrations of E₂ 48 h posttreatment (b = −0.6905; R² = 0.23). In summary, cows treated with cloprostenol had lower concentrations of P₄ for the first 12 h following treatment and subsequently greater concentrations of E₂ compared with dinoprost, although no differences were observed in these 2 PGF_2α analogs for time to complete luteolysis or time to ovulation.     

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows and in Mediterranean buffaloes. Genotype B (GTB) is contagious in dairy cows and may occur in up to 87% of cows of a dairy herd. It was the aim of this study to evaluate genotypes present, clinical outcomes, and prevalence of Staph. aureus in milk samples of primiparous Mediterranean dairy buffaloes. Two hundred composite milk samples originating from 40 primiparous buffaloes were collected from May to June 2012, at d 10, 30, 60, 90, and 150 d in milk (DIM) to perform somatic cell counts and bacteriological cultures. Daily milk yields were recorded. Before parturition until 40 to 50 DIM, all primiparous animals were housed separated from the pluriparous animals. Milking was performed in the same milking parlor, but the primiparous animals were milked first. After 50 DIM, the primiparous were mixed with the pluriparous animals, including the milking procedure. Individual quarter samples were collected from each animal, and aliquots of 1 mL were mixed and used for molecular identification and genotyping of Staph. aureus. The identification of Staph. aureus was performed verifying the presence of nuc gene by nuc gene PCR. All the nuc-positive isolates were subjected to genotype analysis by means of PCR amplification of the 16S-23S rRNA intergenic spacer region and analyzed by a miniaturized electrophoresis system. Of all 200 composite samples, 41 (20.5%) were positive for Staph. aureus, and no genotype other than GTB was identified. The prevalence of samples positive for Staph. aureus was 0% at 10 DIM and increased to a maximum of 22/40 (55%) at 90 DIM. During the period of interest, 14 buffaloes tested positive for Staph. aureus once, 6 were positive twice, and 5 were positive 3 times, whereas 15 animals were negative at every sampling. At 90 and 150 DIM, 7 (17.5%) and 3 buffaloes (7.5%), respectively, showed clinical mastitis (CM), and only 1 (2.5%) showed CM at both samplings. At 60, 90, and 150 DIM, 1 buffalo was found with subclinical mastitis at each sampling. At 30, 60, 90, and 150 DIM, 2.5 (1/40), 22.5 (9/40), 35 (14/40), and 10% (4/40) were considered affected by intramammary infection, respectively. Buffaloes with CM caused by Staph. aureus had statistically significantly higher mean somatic cell count values (6.06 ± 0.29, Log₁₀ cells/mL ± standard deviation) and statistically significantly lower mean daily milk yields (7.15 ± 1.49, liters/animal per day) than healthy animals (4.69 ± 0.23 and 13.87 ± 2.64, respectively), buffaloes with IMI (4.82 ± 0.23 and 11.16 ± 1.80, respectively), or with subclinical mastitis (5.47 ± 0.10 and 10.33 ± 0.68, respectively). Based on our knowledge, this is the first time that Staph. aureus GTB has been identified in milk samples of dairy Mediterranean buffaloes.     

Milk production and enteric methane emissions by dairy cows grazing fertilized perennial ryegrass pasture with or without inclusion of white clover

An experiment was undertaken to investigate the effect of white clover inclusion in grass swards (GWc) compared with grass-only (GO) swards receiving high nitrogen fertilization and subjected to frequent and tight grazing on herbage and dairy cow productivity and enteric methane (CH₄) emissions. Thirty cows were allocated to graze either a GO or GWc sward (n = 15) from April 17 to October 31, 2011. Fresh herbage [16 kg of dry matter (DM)/cow] and 1 kg of concentrate/cow were offered daily. Herbage DM intake (DMI) was estimated on 3 occasions (May, July, and September) during which 17 kg of DM/cow per day was offered (and concentrate supplementation was withdrawn). In September, an additional 5 cows were added to each sward treatment (n = 20) and individual CH₄ emissions were estimated using the sulfur hexafluoride (SF₆) technique. Annual clover proportion (±SE) in the GWc swards was 0.20 ± 0.011. Swards had similar pregrazing herbage mass (1,800 ± 96 kg of DM/ha) and herbage production (13,110 ± 80 kg of DM/ha). The GWc swards tended to have lower DM and NDF contents but greater CP content than GO swards, but only significant differences were observed in the last part of the grazing season. Cows had similar milk and milk solids yields (19.4 ± 0.59 and 1.49 ± 0.049 kg/d, respectively) and similar milk composition. Cows also had similar DMI in the 3 measurement periods (16.0 ± 0.70 kg DM/cow per d). Similar sward and animal performance was observed during the CH₄ estimation period, but GWc swards had 7.4% less NDF than GO swards. Cows had similar daily and per-unit-of-output CH₄ emissions (357.1 ± 13.6 g of CH₄/cow per day, 26.3 ± 1.14 g of CH₄/kg of milk, and 312.3 ± 11.5 g of CH₄/kg of milk solids) but cows grazing GWc swards had 11.9% lower CH₄ emissions per unit of feed intake than cows grazing GO swards due to the numerically lower CH₄ per cow per day and a tendency for the GWc cows to have greater DMI compared with the GO cows. As a conclusion, under the conditions of this study, sward clover content in the GWc swards was not sufficient to improve overall sward herbage production and quality, or dairy cow productivity. Although GWc cows had a tendency to consume more and emitted less CH₄ per unit of feed intake than GO cows, no difference was observed in daily or per-unit-of-output CH₄ emissions.     

Vitamin D₂ impairs utilization of vitamin D₃ in high-yielding dairy cows in a cross-over supplementation regimen

Vitamin D exists in 2 forms that are important regarding vitamin D status and supply in cattle: vitamin D₂ (D₂) and vitamin D₃ (D₃). To become physiologically active, both D₂ and D₃ must undergo 25-hydroxylation in the liver. The resulting 25-hydroxyvitamin D₂ [25(OH)D₂] and 25-hydroxyvitamin D₃ [25(OH)D₃] are measured as indicators of the physiological vitamin D status of cattle. The study used 14 Danish Holstein cows housed without access to sunlight. The cows were orally administered 250 mg (1.0 × 10⁷ IU) of D₂ and D₃ in a cross-over design with 2 treatment groups and 2 study periods, rendering 4 treatments when carryover effects were taken into account: D₂ given first, D₂ given last after D₃, D₃ given first, and D₃ given last after D₂. Two weeks elapsed between the treatment in the first study period and the treatment in the second study period. Blood samples were collected 0, 3, 6, 14, 17, 20, 23, 26, 40, 48, 70, 94, 166, and 214 h after providing the oral bolus of vitamin to the cows. Comparisons between plasma levels of the metabolites D₂, D₃, 25(OH)D₂, and 25(OH)D₃ over time were made by comparing areas under the plasma concentration curves. Oral administration of D₃ increased plasma D₃ (182.6 ± 17.1 ng/mL; mean ± SEM) and 25(OH)D₃ (103.5 ± 10.0 ng/mL) more efficiently than oral administration of D₂ increased plasma D₂ (49.1 ± 32.6 ng/mL) and 25(OH)D₂ (27.9 ± 2.1 ng/mL). The D₃ given after an oral dose of D₂ was less efficient for increasing plasma concentrations of 25(OH)D₃ (61.2 ± 12.0 ng/mL) compared with D₃ given without previous D₂ administration (103.5 ± 10.0 ng/mL), whereas the plasma concentrations of D₃ itself were the same when given first (182.6 ± 17.1 ng/mL) as when given after D₂ (200.0 ± 123.9 ng/mL). The same occurred for plasma concentrations of D₂ metabolites both if D₂ was given first (49.1 ± 32.6 ng/mL) and after D₃ (54.7 ± 7.7 ng/mL). In conclusion, D₃ given after D₂ is less efficient at increasing the plasma status of 25(OH)D₃ than D₃ given without previous D₂ administration.     

The effect of calcium-naloxone treatment on blood calcium, beta-endorphin, and acetylcholine in milk fever

Milk fever is a postpartum syndrome of cows characterized by acute hypocalcemia, which reduces the release of acetylcholine (ACH), inducing flaccid paralysis and recumbency. Our aim was to evaluate the effect of calcium (Ca²⁺) combined with naloxone (Nx, an opioid antagonist; Ca²⁺-Nx) on plasma concentrations of ACH, ß-endorphin (ßE), and Ca²⁺ just before treatment (T0) and at 15, 30, and 90 min after treatment (T15, T30, and T90, respectively). Thirty cows were divided into 3 groups of 10 cows each. In group A1, cows affected by milk fever were treated (i.v.) with a combination of 0.2 mL/kg of body weight (BW) of Ca²⁺ borogluconate (20%) and 0.01 mg/kg of BW of Nx hydrochloride dihydrate. In group A2, cows affected by milk fever were treated (i.v.) with 2 mL/kg of BW of Ca²⁺ borogluconate (20%). In group C, healthy cows were treated (i.v.) with a combination of 0.2 mL/kg of BW of Ca²⁺ borogluconate (20%) and 0.01 mg/kg of BW of Nx hydrochloride dihydrate. Cows underwent treatments within 24 h of calving. Blood samples were collected at T0 and at T15, T30, and T90 for quantitative determination of ACH, ßE, and Ca²⁺. The cows in groups A1 and A2 recovered within a mean of 20 ± 10 min, although 4 cows in group A2 underwent a relapse. Blood Ca²⁺ concentrations in group C increased slightly at T30 and at T90 (T30: 8.8 ± 0.6 mg/dL; T90: 8.7 ± 0.6 mg/dL) after treatment, whereas the response in groups affected by milk fever was similar, even though Ca²⁺ concentrations showed a sharp increase (A1: 8.9 ± 0.8 mg/dL; A2: 6.0 ± 0.7 mg/dL), particularly at T15 in group A1. Concentrations of ßE showed a similar pattern in groups A1 and C, with an increase at T15 (A1: 8.2 ± 1.0 ng/mL; C: 2.7 ± 0.4 ng/mL) and a subsequent decrease until T90 (A1: 1.4 ± 0.3 ng/mL; C: 1.4 ± 0.4 ng/mL), whereas ßE remained constant throughout in group A2. Concentrations of ACH in group A1 decreased significantly between T0 and T15, T30, and T90 (T0: 7.2 ± 1.1 nmol/L; T15: 4.2 ± 1.2 nmol/L; T30: 2.9 ± 0.8 nmol/L; T90: 3.1 ± 0.3 nmol/L), whereas in group A2, it did not change. In group C, concentrations of ACH decreased at T15 and increased again at T30 (T15: 1.1 ± 0.3 nmol/L; T30: 3.2 ± 0.7 nmol/L). Our results suggest that administration of Ca²⁺-Nx, which restored the physiological Ca²⁺ concentrations, might have an effect on nicotinic receptors by restoring the normal neuromuscular transmission at the motor endplate.     

Effects of short-term glucagon administration on gluconeogenic enzymes in the liver of midlactation dairy cows

During lactation, the dairy cow experiences an increased demand for glucose to support milk production. Increased glucose demand can be met through increased capacity for gluconeogenesis, increased supply of glucose precursors, or a combination of both processes. Glucagon, a key hormone in glucose homeostasis, acts to promote gluconeogenesis and increase glucose output from liver. The objective of this study was to determine the effect of short-term administration of glucagon on expression of gluconeogenic enzymes in lactating dairy cattle. Sixteen multiparous Holstein cows were selected from the Purdue University Animal Sciences Dairy Research Center herd. Cows were stratified on the basis of milk production and days in milk and randomly assigned to either a saline or glucagon injection group (n = 8 per group). Cows were injected subcutaneously at −21, −14, −7, and 0 h relative to final glucagon and saline injections with either 3.75 mg of lyophilized bovine glucagon (15 mg/d) dissolved in 60 mL of 0.15 M NaCl (pH 10.25) or 60 mL of 0.15 M NaCl. Liver biopsy samples were obtained 1 wk before injection to establish baseline values and at 3 h after cows received final glucagon and saline injections. Biopsy samples were analyzed for mRNA abundance, enzyme activity, protein abundance, and in vitro measures of gluconeogenesis. Glucagon did not alter pyruvate carboxylase or cytosolic phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance, enzyme activity, or protein abundance, although there was a tendency for greater mRNA expression with the glucagon treatment (4.69 vs. 6.78, arbitrary units). Glucagon injections did not change mitochondrial PEPCK mRNA expression. Gluconeogenesis from 2.5 mM [2-¹⁴C]propionate and 2.0 mM [U-¹⁴C]lactate was similar in liver biopsy samples from glucagon-treated and control cows. There was no effect of glucagon on dry matter intake and milk production. Glucose, nonesterified fatty acids, β-hydroxybutyrate acid, and insulin were not altered by glucagon. Blood glucagon was elevated, 76.09 vs. 96.14 pg/mL, for cows receiving glucagon injections. The data indicate that 24-h administration of glucagon does not alter cytosolic PEPCK mRNA expression or result in immediate alterations in total PEPCK enzyme activity and gluconeogenic capacity.     

Presynchronization using a modified Ovsynch protocol or a single gonadotropin-releasing hormone injection 7 d before an Ovsynch-56 protocol for submission of lactating dairy cows to first timed artificial insemination

Presynchronization strategies, such as Presynch-Ovsynch and Double-Ovsynch, increase fertility to timed artificial insemination (TAI) compared with Ovsynch alone; however, simpler presynchronization strategies could reduce costs and simplify reproductive management. Lactating Holstein cows (n = 601) were randomly assigned to 1 of 2 presynchronization treatments before beginning an Ovsynch-56 protocol (GnRH at 70 ± 3 DIM, PGF_2α 7 d later, GnRH 56 h after PGF_2α, and TAI 16 h later at 80 ± 3 DIM) for first TAI. Cows (n = 306) in the first treatment (Double-Ovsynch; DO) were presynchronized using a modified Ovsynch protocol (GnRH at 53 ± 3 DIM, 7 d later PGF_2α, and GnRH 3 d later) ending 7 d before the first GnRH injection (G1) of an Ovsynch-56 protocol. Cows (n = 295) in the second treatment (GGPG) were presynchronized using a single injection of GnRH 7 d before G1 of an Ovsynch-56 protocol at 63 ± 3 DIM. Blood samples were collected at G1 and the PGF_2α injections of the Ovsynch-56 protocol to determine progesterone (P4) concentrations. Pregnancy diagnosis was performed using ultrasonography 32 d after TAI, and pregnant cows were reexamined 46 and 70 d after TAI. Overall, DO cows had more pregnancies per artificial insemination (P/AI) compared with GGPG cows 32 d after TAI (53 vs. 43%). Overall, P/AI did not differ by parity (primiparous vs. multiparous), and pregnancy loss did not differ between treatments or parities. More DO cows had P4 in a medium range (>0.5 to <4 ng/mL) at G1 of the Ovsynch-56 protocol compared with GGPG cows (82 vs. 50%), and more DO cows had high P4 (>4 ng/mL) at the PGF_2α injection of the Ovsynch-56 protocol compared with GGPG cows (67 vs. 36%). Thus, presynchronization with a modified Ovsynch protocol increased P/AI after TAI at first AI by increasing synchrony to the Ovsynch-56 protocol compared with presynchronization using a single injection of GnRH.     

Body temperature around induced estrus in dairy cows

The overall objective of this study was to study the influence of induced estrus on body temperature, comparing 5 distinct intervals around induced estrus and to determine the diurnal pattern from 4 ± 1 d before to 4 ± 1 d after induced estrus. Sixteen estrous cycles of 9 postpartum dairy cows were synchronized with 2 injections of PGF_2α, 10 d apart. After the second PGF_2α injection on d 10, temperature loggers were inserted into the vaginal cavity for a 12 ± 1-d period. Two days later, a third dose of PGF_2α was injected to induce estrus. After confirmation of a corpus luteum, loggers were removed on d 5 ± 1. Observation of estrus, rectal palpation, and ultrasound scanning to determine ovulation were carried out every 4 ± 1 h, beginning at 12 h after the third PGF_2α injection. Blood samples from the vena coccygea mediana were collected twice daily from d 11 to 12 and every 4 ± 1 h after the third PGF_2α injection until ovulation. Vaginal temperature was recorded every 5 min and averaged to hourly means for the following 5 periods: 1) 48 h preceding the third PGF_2α injection, 2) from the third PGF_2α injection to first signs of estrus, 3) estrus to ovulation, 4) a 4-h interval in which ovulation occurred, and 5) a 96-h post-ovulation period. High body temperatures (39.0 ± 0.5°C) and low progesterone (P4) concentrations (<0.5 ng/mL) were observed during estrus, whereas low body temperatures were observed from PGF_2α injection to estrus (38.6 ± 0.3°C) and around ovulation (38.5 ± 0.2°C), respectively. An association between body temperature and serum P4 concentrations did not exist. However, P4 concentrations on d 11 and 12 were high (5.0 ± 1.5 ng/mL) and decreased (0.9 ± 0.2 ng/mL) after ovulation. Diurnal temperature rhythms were similar before and after estrus. Vaginal temperature before estrus (d 11 and 12) was slightly (0.1°C) higher compared with the post-ovulation period.     

Effects of twice-daily nursing on milk ejection and milk yield during nursing and milking in dairy cows

Milk production and hormonal responses to milking in Holstein cows that were milked twice daily, and that either also nursed calves twice daily 2 h after milking for 9 wk after calving (n = 10) or that served as nonnursing controls (n = 8) were examined to assess how nursing affected responses to machine milking. Milk yield at milking during the 9 wk of nursing was lower in nursing cows compared with control cows (26.1 ± 1.0 vs. 35.5 ± 1.1 kg) that were only machine milked. During nursing, the amount drunk by calves increased from 6.5 ± 0.7 kg/d on wk 1 to 12.5 ± 1.4 kg/d on wk 9. When this was added to the amount of milk obtained at milking, nursing cows did not differ from control cows in total milk produced (35.5 ± 1.0 vs. 35.5 ± 1.0 kg). Residual milk yield, after i.v. injection of oxytocin after milking, was higher in nursing cows than in control cows (8.7 ± 0.8 vs. 3.2 ± 0.8 kg). During the 6 wk after weaning, milk production was the same for the nursing and control cows (34.0 ± 1.35 vs. 34.7 ± 1.42 kg). Plasma oxytocin levels during milking were greater for control cows than for nursing cows (31.7 ± 5.4 vs. 18.0 ± 2.8 pg/mL), but were equivalent to concentrations in nursing cows during nursing (35.5 ± 7.5 pg/mL). Plasma concentrations of prolactin and cortisol increased after both milking (control vs. nursing: prolactin: 40.2 ± 6.8 vs. 32.9 ± 6.1 ng/mL; cortisol: 6.4 ± 1.23 vs. 7.4 ± 1.10 ng/mL) and nursing (control vs. nursing: prolactin: 18.6 ± 7.3 vs. 38.9 ± 6.6 ng/mL; cortisol: 2.34 ± 1.15 vs. 7.37 ± 1.04 ng/mL). In contrast to previous studies, there was no obvious advantage for milk production by keeping a calf with the cow. This appears to result from the reduced oxytocin secretion during milking for the nursing cows.