Serum concentration and mRNA expression in milk somatic cells of toll-like receptor 2, toll-like receptor 4, and cytokines in dairy cows following intramammary inoculation with Escherichia coli

The objective of the current study was to investigate the toll-like receptors (TLR), including the soluble forms sTLR2 and sTLR4, involved in innate immune responses of dairy cows to experimentally induced Escherichia coli mastitis. Six clinically healthy Holstein dairy cows received an intramammary inoculation of E. coli O111:K58 between 63 and 83 d postpartum. Concentrations of sTLR2 and sTLR4, the proinflammatory cytokines IL-6 and tumor necrosis factor-α (TNF-α), and acute phase proteins serum amyloid A (SAA) and haptoglobin (Hp) in blood were measured by ELISA. Furthermore, 10 mL of milk was collected from challenged quarters immediately before inoculation and at 6, 12, 24, 48, and 72 h after inoculation, and mRNA expression of selected genes, including TLR2, TLR4, IL-1β, IL-6, TNF-α, and IL-8, was quantified by real-time PCR. Escherichia coli intramammary infection elicited a decrease in the circulating levels of leukocytes. Rectal temperature was elevated at 6 h postinoculation (PI). Similarly, the serum concentrations of TNF-α, IL-6, and SAA increased at 6 h PI. However, serum concentrations of sTLR2, sTLR4, and Hp did not differ after challenge. The mRNA expression of TLR2, IL-1β, and IL-8 in milk somatic cells increased at 12 h PI, whereas a decreased IL-6 mRNA expression was detected from 6 to 48 h PI. In conclusion, we found that TLR2 mRNA expression increased in milk somatic cells collected from infected quarters of cows challenged with E. coli, whereas the concentrations of sTLR2 and sTLR4 remained unchanged after challenge. Thus, sTLR2 and sTLR4 may protect the host by sequestrating pathogen-associated molecular patterns during E. coli mastitis.     

Determination of milk and blood concentrations of lipopolysaccharide-binding protein in cows with naturally acquired subclinical and clinical mastitis

Blood and milk concentrations of the acute phase protein lipopolysaccharide-binding protein (LBP) were evaluated in cows with naturally occurring mastitis. Blood and milk samples were collected from 101 clinically healthy dairy cows and 17 dairy cows diagnosed with clinical mastitis, and the LBP concentrations of the samples were measured by an ELISA. Concentrations of LBP were greater in the blood and milk of cows with clinical mastitis than in those with healthy quarters. Concentrations of LBP also differed between uninfected and subclinically infected quarters with low somatic cell count. Blood concentrations of LBP in cows with subclinical intramammary infections could not be differentiated from those of cows with all healthy quarters. Together, these data demonstrate that increased blood and milk concentrations of LBP can be detected in dairy cows with naturally acquired intramammary infections that cause clinical mastitis.     

Association of time to metritis diagnosis with circulating concentration of metabolites,minerals, and haptoglobin in Jersey cows

Our objective was to investigate the association of early metritis [EMET, diagnosed at <5 d in milk (DIM)] and late metritis (LMET, diagnosed at ≥5 DIM) with circulating concentrations of energy metabolites, minerals, and haptoglobin (Hp) throughout the first 14 d postpartum. A total of 379 purebred Jersey cows were enrolled in a prospective cohort study from a single herd in west Texas. Cows were examined for metritis using the Metricheck device (Simcro Ltd.) at 4, 7, and 10 DIM. Cows identified by farm employees as possible metritis cases were also evaluated for metritis. Blood samples were collected for analysis of concentrations of Ca, Mg, and glucose at DIM 1 through 5, 7, 10, and 14. Albumin, urea, fructosamine, free fatty acids (FFA), creatinine, and β-hydroxybutyrate (BHB) were analyzed at DIM 3, 5, 7, 10, and 14, and Hp at DIM 1 through 5 and 7. Data were analyzed using the MIXED and PHREG procedures of SAS (SAS Institute Inc.). A series of mixed general linear models accounting for repeated measures were fitted to the data. The independent variables metritis [no metritis (NMET), EMET, and LMET], DIM of analyte assessment, and parity were forced in all models. Multivariable Cox proportional hazard models were built to assess the risk of pregnancy and culling within 150 DIM. The overall metritis incidence was 26.9% (EMET = 49; LMET = 53; NMET = 277). Average concentrations of glucose, Mg, and urea were not associated with metritis. The associations of Ca, creatinine, BHB, and fructosamine with metritis were dependent on the DIM of analyte assessment. Cows categorized as EMET and LMET had, on average, lower albumin and fructosamine compared with NMET cows. Both EMET and LMET cows had, on average, greater BHB than NMET cows. A greater FFA concentration was only observed in cows diagnosed with EMET compared with NMET cows (EMET = 0.58, LMET = 0.52, NMET = 0.48 mmol/L). Additionally, circulating Hp concentration was greater for LMET and EMET compared with NMET cows, and EMET cows had greater Hp compared with LMET cows (EMET = 1.15; LMET = 1.00; NMET = 0.84). In conclusion, several blood biomarkers were temporally associated with early versus late metritis diagnosis in postpartum Jersey cows. No meaningful differences were observed in production, reproduction, or culling between EMET and LMET cows. These results suggest that cows with EMET undergo a more severe degree of inflammation and negative energy balance compared with NMET cows.     

Production effects of extruded soybean meal replacing canola meal in the diet of lactating dairy cows

This study investigated the effects of extruded soybean meal (ESBM) in comparison with canola meal (CM) fed on an equivalent crude protein (CP) basis on lactational performance and ruminal fermentation of dairy cows. Following a 2-wk covariate period, 48 Holstein cows averaging (±SD): 146 ± 46 d in milk (DIM) and 43 ± 7 kg/d milk yield (MY) were assigned 1 of 2 treatment diets in a randomized complete block design experiment, which included a 2-wk period for dietary treatment adaptation before experimental data were collected. Following the adaptation period, samples and experimental data were collected for a total of 7 wk. Cows were blocked based on parity, DIM, and MY. Treatment diets contained 15.8% CM (containing 41.2% CP) or 13.2% ESBM (with 48.7% CP) of total mixed ration dry matter (DM), with similar inclusion of other feed ingredients. The CM diet was supplemented with canola oil, whereas the ESBM diet was supplemented with soybean hulls to achieve similar ether extract and neutral detergent fiber contents between the diets. Urea and rumen-protected Met and Lys were added to both diets to meet or exceed cow recommendations. Whole-ruminal digesta samples were collected from 10 (5 per treatment) ruminally cannulated cows. Eight cannulated cows were removed during the last week of the experiment to participate in another study. Treatment did not affect DM intake and MY or energy-corrected MY of the cows. Energy-corrected MY, apart from experimental wk 5, was similar between treatments. Apart from experimental wk 3 and 7, milk fat concentration and yield were greater for cows fed ESBM compared with CM. In multiparous cows only, milk true protein yield was greater for cows fed CM compared with ESBM. Ruminal concentration of total volatile fatty acids and the molar proportion of acetate were greater for ESBM, and propionate and valerate were greater in cows fed CM. Acetate to propionate ratio was greater for cows fed ESBM versus CM diet. Compared with the CM diet, the ESBM diet increased plasma concentrations of Ile, Leu, and Phe but not the sum of essential AA. Apparent total-tract digestibility of acid detergent fiber was greater in cows fed ESBM relative to CM. In this experiment, CM and ESBM included on an equal CP basis in the diet of dairy cows, resulted in similar DM intake, MY, and feed efficiency.     

Associations of neonatal acute phase response with first lactation performance in dairy cows

During the first 3 wk of life, the immune system of newborn ruminants starts to work, as indicated by fluctuations in the concentrations of proinflammatory cytokines and acute phase proteins (APP). They have been shown to be markers for short and long-term weight gain in ruminants. This observational study investigated these proteins as possible indicators of first lactation performance of dairy cows. A total of 117 dairy calves from a single farm were enrolled in the study. Serum and fecal samples were taken once a week for the first 3 wk of life. Cryptosporidium spp. infection and its treatment were monitored and accounted for in statistical analysis. The concentrations of the APP serum amyloid A (SAA) and haptoglobin (Hp), and the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were analyzed in serum. After the first lactation, health and performance data of the cows were retrieved, and associations between APP and cytokine concentrations with first lactation outcomes were investigated through linear and logistic regression. This study found a negative association between the concentration of Hp in the second week of life and average daily weight gain at one year. The SAA concentration measured during the second and third weeks of life was positively associated with age at first calving. IL-6, SAA, and Hp measured in the second week of life were positively associated with calving to conception interval. The concentrations of IL-6 and TNF-α during the first week of life were associated with higher odds of reproductive issues during the first lactation period. These markers can be used to help understand underlying processes that influence animal health and production. In conclusion, inflammatory responses during the first week of life are related to reproductive success, whereas the second and third weeks of life seem to influence the future productive performance in dairy cows.     

Reducing milking frequency from thrice to twice daily in early lactation improves the metabolic status of high-yielding dairy cows with only minor effects on yields

Reducing milk production during early lactation might be of interest to improve the energy balance (EB) of high-yielding dairy cows. Therefore, the objective of this study was to determine how reducing the milking frequency (MF) of high-yielding dairy cows from thrice to twice a day during the first 30 d in milk (DIM) affects yields, intake, efficiency, metabolic status, and carryover effects. To this end, 42 multiparous cows were divided into 2 groups according to their previous lactation performance, parity, and body weight. The control cows were milked 3 times a day (3ML) and the treated cows were milked twice a day (2ML) until 30 DIM and then both groups were milked 3 times a day. Milk samples were taken twice a week from 2 or 3 consecutive milkings until 45 DIM for analysis of milk solids, and both groups were followed until 100 DIM to determine the carryover effects of MF until 30 DIM. Individual dry matter intake (DMI), milk yield, and body weight were recorded daily. Blood samples were taken 3 times weekly from 14 d prepartum until 45 DIM. Milk yield during the first 30 DIM was 8.6% higher (49.3 and 45.4 kg/d, respectively), milk fat percentage was lower (3.96 and 4.27%, respectively), and the yields of all milk solids were higher in the 3ML cows than in the 2ML cows. Dry matter intake and 4% fat-corrected milk were similar between groups. The EB during the first 30 DIM was lower in the 3ML cows than in the 2ML cows, and milk yield, but not 4% fat-corrected milk yield, per unit of DMI was higher in the 3ML cows. No differences were observed between groups from 31 to 100 DIM in milk yield (～56.3 kg/d for both groups), milk solids yield, DMI, or milk/DMI; however, fat percentage was lower and EB was higher in the 3ML cows. Blood glucose concentrations between 0 and 30 DIM were lower and β-hydroxybutyrate concentrations were higher in the 3ML cows than in the 2ML cows, but nonesterified fatty acids concentrations were lower, which may be attributed to the lower clearance frequency of nonesterified fatty acids from the blood stream in the 2ML cows. A lower proportion of the 3ML cows (10%) ovulated ≤15 DIM compared with the 2ML cows (40%), with no beneficial effects on preovulatory follicle characteristics. Reducing the MF from thrice to twice a day during the first 30 DIM improved EB and metabolic status, with only minor effects on production.     

Effect of selenium source on selenium status, neutrophil function, and response to intramammary endotoxin challenge of dairy cows

The effects of feeding dry and early lactation dairy cows diets with selenate or selenized yeast (Se-yeast) on concentrations of Se in serum, milk, and newborn calves, neutrophil function, and inflammatory response were determined. At 60 d before anticipated calving until approximately 30 d in milk (DIM), cows were fed diets that contained 0.3 mg of supplemental Se/kg of DM from sodium selenate or Se-yeast. Diets also contained 0.2% supplemental S (as sulfate) because it has been shown to reduce absorption of Se by dairy cows. The concentration of Se in serum at calving and 28 DIM was about 1.4 times greater for cows fed Se-yeast than for those fed selenate. Serum concentrations decreased 45 and 23% from dry-off to calving for cows fed selenate or Se-yeast, respectively. Selenium concentrations in serum from newborn calves were also about 1.4 times greater when the dams were fed Se-yeast. Concentrations of Se in colostrum and milk were about 1.8 times greater when cows were fed Se-yeast. Blood neutrophils were isolated from cows at 28 DIM and were used in an in vitro kill assay. Selenium treatment did not affect bacterial kill or the percentage of neutrophils that phagocytized bacteria. At approximately 28 DIM, one quarter from each cow was infused with a solution containing endotoxin. Peak body temperature (40.7°C) occurred 6 h postinfusion, and peak somatic cell count (6.5 log₁₀/mL) occurred at 12 h postinfusion. Neither measure was influenced by Se treatment.     

Dietary-induced negative energy balance has minimal effects on innate immunity during a Streptococcus uberis mastitis challenge in dairy cows during midlactation

Ten multiparous Holstein cows were used to determine the effects of negative energy balance (NEB) on the immune response to a Streptococcus uberis (strain O140J) mastitis challenge during midlactation. Before the study, milk from all quarters of each cow was bacteriologically negative, with a composite somatic cell count of <200,000 cells/mL. Cows were paired based on parity, days in milk, and milk yield. At approximately 77 d in milk, half the cows (n = 5) were feed-restricted to 60% of calculated net energy for lactation requirements to induce NEB. Feed restriction lasted 7 d. Control cows (n = 5) were fed the same diet ad libitum (i.e., positive energy balance; PEB). After 5 d, one rear quarter in all cows was inoculated with 5,000 cfu of Strep. uberis. Jugular blood and aseptic quarter milk samples were collected daily until inoculation and every 6 h postinoculation for 36 h. Blood was analyzed for nonesterified fatty acids, β-hydroxybutyrate, insulin, cortisol, albumin, serum amyloid A (SAA), and haptoglobin (Hp). Periodically throughout the trial period, blood neutrophils were isolated for determination of cell morphology, chemotaxis, and phagocytosis capability in vitro. Quarter milk samples were analyzed for concentrations of SAA, Hp, cytokines (tumor necrosis factor-α, IL-10 and IL-1β), and activity of respiratory burst enzymes (superoxide dismutase and glutathione peroxidase). All cows developed local and systemic signs of mastitis and calculated NEB was similar to that of cows experiencing postpartal NEB. Serum glucose and insulin concentrations increased in both groups after challenge, most likely because of enhanced glycogenolysis and gluconeogenesis; results indicate that immune cell function may be glucose dependent. Serum cortisol concentration was higher in NEB than PEB cows during feed restriction only (before inoculation), and serum albumin concentration was higher in NEB than PEB cows during the infection period. Compared with PEB, cows in NEB had lower SAA concentrations in serum after 5 d of feed restriction but higher SAA concentrations in milk after Strep. uberis challenge. Serum Hp concentration was higher by 36 h postchallenge in NEB than in PEB cows. Phagocytic capability of neutrophils was lower in NEB than in PEB cows at 0 h of infection but decreased in both PEB and NEB cows through 36 h postinfection. Our results indicate that cows subjected to dietary-induced NEB during midlactation had relatively minimal alterations in immune function.     

Mare's and cow's milk: promote similar metabolic effects and expression of innate markers in Caco-2 cells?

Mare's milk (MM) is very similar to human breast milk, as it consists of low concentrations of proteins and fat and high concentrations of lactose. Thus, MM can be considered as an alternative to cow's milk (CM) for human nutrition. In this study, proteolytic resistant peptides to gastrointestinal enzymes were measured by reverse phase-high-performance liquid chromatography (RP-HPLC-PDA-UV) to determine their relative uptake by intestinal epithelial (Caco-2) cells. Alterations in mitochondrial enzyme activities (MTT test), inner mitochondrial membrane potential (ΔΨm), cell cycle progression and the expression (mRNA) of inflammatory (IL-1β, TNF-α and NF-κB) markers were studied. This is the first study comparing the effects of proteolytic resistant peptides from the whey proteins of mare's milk to those originating from cow's milk on intestinal cells. Similar proteolytic resistant peptide patterns, but lower uptake rates, were observed for MM peptides (7–40%) compared to peptides from CM (40–60%), although both types of peptides, probably derived from α-lactalbumin, were still capable of impairing ΔΨm in intestinal cells (CM > MM).Both MM and CM increased the expression of TNF-α, whereas IL-1β levels were not affected. CM increased NF-κB mRNA levels. Overall, our data indicate that proteolytic resistant peptides from both MM and CM exert similar metabolic effects on mitochondrial metabolism (up to 30%) in intestinal Caco-2 cells. However, the low uptake rates of peptides from MM may be beneficial to the human diet, an alternative to CM. Further in vivo studies are needed to better understand the potential clinical relevance of the control and/or regulation of metabolic processes related to perturbations in the gut immune system associated with lymphoid tissue.     

Milk production change following clinical mastitis and reproductive performance compared among J5 vaccinated and control dairy cattle

Naturally occurring cases of bovine clinical mastitis (CM) were studied among J5 vaccinates and controls on 3 commercial dairy farms. Milk production change and reproductive performance following CM were compared between the 2 groups. Among 306 controls and 251 vaccinates, there were 221 new cases of CM affecting 120 cows; 437 lactations never had a case of CM. Environmental pathogens made up 90% (159/176) of etiologic agents isolated. Change in daily milk production following CM was associated with J5 vaccination, days in milk (DIM) at onset of CM, and herd effect as well as each 2-way interaction between the 3 factors. The adjusted daily milk for 21 d following CM was 7.6 kg greater among J5 vaccinates than controls; however, this protective effect of vaccination waned with increasing DIM at onset of CM. A mixed linear model with autoregressive order 1 [AR(1)] correlation structure estimated the daily milk production of any cow (whether or not she had CM) on a given DIM. Cows with CM caused by nonagalactiae streptococci, Staphylococcus aureus, Escherichia coli, or Klebsiella lost significant daily milk production for the entire lactation relative to nonmastitic cows. Another mixed linear model for only coliform CM cases (E. coli, Klebsiella, and Enterobacter) within the first 50 DIM showed milk loss for 21 d following coliform CM to be significantly less for J5 vaccinates than for controls, by 6 to 15 kg per day. Cows were significantly less likely to become pregnant if they had CM caused by E. coli (42% pregnant) or Streptococcus spp. (38% pregnant), whereas 78% (342/437) of cows with no mastitis conceived. Days open (number of days from calving until pregnancy) averaged 131 d for cows with no CM and 162 d for cows that had at least one case of CM. Days until conception, days until last breeding, days open, times bred, and percentage of cows pregnant by 200 DIM were not changed with J5 vaccination. Nonetheless, an important benefit of the use of J5 bacterin appears to be reduction of the loss of daily milk production following CM, whether all cases or only those caused by coliform bacteria were considered.     

The relationship of excessive energy deficit with milk somatic cell score and clinical mastitis

Periparturient cows go through a period of immune suppression often marked by immune cell dysfunction. Further exacerbation of this dysfunction through early-lactation excessive energy deficit (EED) has been associated with increased susceptibility to infectious conditions such as mastitis. Our objective was to explore the association of milk somatic cell score (SCS) and clinical mastitis (CM) diagnosis in cows identified with EED, diagnosed using each of the following: blood and milk β-hydroxybutyrate (BHB), milk predicted blood nonesterified fatty acid (mpbNEFA) concentrations, or milk de novo fatty acid (FA) relative percentages (rel %). We analyzed data collected from 396 multiparous Holstein cows from 2 New York farms in a prospective cohort study. Coccygeal vessel blood samples and composite milk samples were collected twice weekly from 3 to 18 days in milk (DIM) for a total of 4 time points per cow (T1, T2, T3, T4). Blood was analyzed using a hand-held meter, and milk was analyzed using Fourier-transform mid-infrared spectrometry for milk BHB and mpbNEFA concentrations, milk de novo FA rel %, and somatic cell count. Excessive energy deficit was diagnosed as blood BHB ≥ 1.2 mmol/L, milk BHB ≥ 0.14 mmol/L, mpbNEFA ≥ 0.55 mmol/L, or de novo FA ≤ 22.7 rel %, depending on the model. Clinical mastitis cultures were collected from 4 to 60 DIM by on-farm personnel. Incidence of hyperketonemia as determined by blood BHB was 13.4%, and incidence of CM was 23.9%. Separate repeated-measures ANOVA models were developed for each EED diagnostic analyte for parity groups 2, 3, and ≥4 to assess differences in SCS; t-test analyses were similarly used to assess the association of each diagnostic analyte with CM at each time point. For all diagnostic analytes, apart from milk BHB, cows diagnosed with EED tended to have lower SCS than their non-EED counterparts. This was especially apparent at T1 for all parity groups, and at T2, T3, and T4 for blood BHB and mpbNEFA. For EED diagnosis via mpbNEFA, mean SCS were lower in parity ≥4, with a difference in mean SCS between EED and non-EED animals of 0.7 SCS units, equating to a somatic cell count in EED animals approaching half that of non-EED (EED = 67,000 cells/mL, non-EED = 107,000 cell/mL). No important relationships were observed between CM diagnosis and blood BHB, milk BHB, or mpbNEFA. For de novo FA rel %, reductions in this analyte were noted before CM diagnosis at all time points. Although the relationship between EED and CM is still unclear, our findings suggest that cows in EED, diagnosed using blood BHB or mpbNEFA during the first 18 DIM, have a tendency toward lower SCS compared with their non-EED counterparts.     

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.     

Type of cottonseed and level of gossypol in diets of lactating dairy cows: effects on lactation performance and plasma gossypol

Objectives were to determine the effects of feeding whole linted Upland (WUP) and cracked Pima cottonseed (BUPCP) to lactating cows on plasma gossypol concentrations and lactation performance. Holstein cows (n = 813) from three commercial dairies were assigned to one of two diets starting at 13+/-11 d in milk (DIM) for a 170-d experimental period. Cottonseed was included at 10% of the diet dry matter, and treatments consisted of replacing WUP with a 1:2 blend of BUPCP. Blood was collected from all cows at 61 and 91 DIM and from a subset of 56 cows on one dairy at 10, 33, 61, 91, 120, and 152 DIM. Intakes of free gossypol increased 32% in cows receiving the BUPCP diet, and concentrations of total gossypol (TG), as well as the proportion of TG as minus (-) isomer in plasma, were higher for cows fed BUPCP than WUP. Plasma TG increased linearly with increasing DIM, but cows fed the BUPCP, especially multiparous cows, had a higher increase in plasma TG over time. Yields of milk and milk components did not differ between the two treatments, although, yields of milk and milk components were greater in cows with higher plasma TG. Replacement of WUP cottonseed with BUPCP cottonseed increased plasma gossypol, but dry matter intake and yields of milk and milk components were not affected.     

Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cows: II. Endometrial gene expression related to maintenance of pregnancy

The objectives were to examine the effects of bovine somatotropin (bST), pregnancy, and dietary fatty acids on expression of key endometrial genes and proteins regulating prostaglandin synthesis in lactating dairy cows. Two diets were fed, at about 17 d in milk (DIM), in which oil of whole cottonseed (control diet) was compared with calcium salts of fish oil-enriched lipid (FO). Ovulation was synchronized in cows with a presynchronization plus Ovsynch protocol and cows were inseminated artificially or not inseminated on d 0 (d 0 = time of synchronized ovulation; 77 ± 12 DIM). On d 0 and 11, cows received bST (500 mg) or no bST, and were slaughtered on d 17 to recover uterine secretions and endometrial tissue. Number of cows in the control diet: 5 bST-treated cyclic (bST-C), 5 non-bST-treated cyclic (no bST-C), 4 bST-treated pregnant (bST-P), and 5 non-bST-treated pregnant (no bST-P) cows and in the FO diet: 4 bST-treated FO-cyclic (bST-FO-C) and 5 non-bST-treated cyclic (no bST-FO-C) cows. The FO diet increased progesterone receptor (PR) mRNA, and treatment with bST increased PR mRNA concentration in endometrium of no bST-C, but not in no bST-FO-C or no bST-P cows. Concentrations of estrogen receptor-α (ERα) mRNA and protein, and oxytocin receptor (OTR) mRNA were decreased in no bST-P cows compared with no bST-C cows. Treatment with bST tended to increase OTR and ERα mRNA concentrations in cyclic cows fed control or FO diets. Immunohistochemistry demonstrated effects of bST, FO, and pregnancy on distributions of ERα and PR proteins in endometrium. Pregnancy and FO feeding decreased ERα abundance in luminal epithelium. Prostaglandin H synthase-2 (PGHS-2) protein was elevated in pregnant cows and localized to the luminal epithelium. Both FO and bST treatments reduced staining intensity of PGHS-2 protein. Concentrations of prostaglandin E synthase mRNA were elevated in either cyclic or pregnant cows in response to bST, whereas bST decreased prostaglandin F synthase mRNA in pregnant cows. Uterine lumen fluids had more PGF_2α and prostaglandin E₂ in pregnant than cyclic cows. Uterine lumen fluids of bST-P cows contained more prostaglandin E₂ than those from no bST-P cows. In summary, both pregnancy and bST altered endometrial gene expression, and cyclic cows responded differently to bST than pregnant cows. Feeding FO modulated PR, ERα, and PGHS-2 expression and distribution among endometrial cell types in a manner that may favor establishment and maintenance of pregnancy.     

柚叶总黄酮对LPS所致Caco-2结肠上皮细胞间高通透性的保护作用

探讨柚叶总黄酮(PLTFE)对脂多糖(LPS,2 μg/mL)诱发Caco-2高通透性细胞模型的保护作用。Caco-2模型细胞以不同浓度的PLTFE(0、10、50、100、150 μg/mL)处理培养24 h进行后续实验。MTT法测定细胞生存率,细胞乳酸脱氢酶(lactate dehydrogenase,LDH)水平依说明书使用试剂盒测定。酶联法(enzyme linked immunosorbent assay,ELISA)测定白介素(interlukin-1β,IL-1β)、IL-8和肿瘤坏死因子(tumor necrosis factor-α,TNF-α)分泌水平。跨上皮细胞电阻(trans epithelial electrical resistance,TEER)值和异硫氰酸荧光素-右旋糖酐(FD40)透过度用于评估细胞通透性水平。实时定量PCR(Quantitative real-time PCR,qRT-PCR)检测细胞IL-1β、IL-8、TNF-α、闭锁蛋白(Occludin)、紧密连接蛋白-1(claudin-1)、封闭小带蛋白(ZO-1)和肌球蛋白轻链激酶(myosin light chain kinase,MLCK)的mRNA表达。结果表明,柚叶总黄酮能显著提高受损细胞生存率至86.1%,抑制受损细胞中LDH的溢出(p<0.05)。同时还能有效抑制受损细胞中炎性细胞因子(IL-1β、IL-8、TNF-α)的分泌及mRNA转录。此外,柚叶总黄酮可增强细胞紧密连接因子(Occludin、claudin-1、ZO-1)的mRNA转录,抑制MLCK的mRNA转录,改善细胞间高通透性qRT-PCR法检测细胞中相关因子的mRNA转录水平。结果提示,柚叶总黄酮具有较强的抗炎活性,能通过上调细胞内细胞紧密连接相关因子的mRNA转录显著改善LPS造成的Caco-2细胞间高通透性的发生(p<0.05)。     

Direct-fed microbial supplementation and health and performance of pre- and postpartum dairy cattle: a field trial

A double-blind field trial was conducted on a commercial dairy to study the effects of feeding a direct-fed microbial (DFM) product consisting of 2 strains of Enterococcus faecium plus Saccharomyces cerevisiae yeast on prepartum and postpartum performance of Holstein cows. Treatments consisted of the normal pre- and post-fresh TMR supplemented with the DFM (2 g/cow per d) or a placebo. Treatments started approximately 10 d prepartum and continued until about 23 d in milk (DIM). A total of 366 Holstein cows were enrolled in 1 of 2 placebo groups or 2 DFM-supplemented groups. Groups were enrolled consecutively, starting with the placebo treatment. Sample size was limited to 4 groups because the cooperating dairy prematurely terminated the study due to increased health problems in one of the groups. Blood samples were taken during the prefresh period between 2 and 10 d prior to calving and at weekly intervals from 3 to 23 DIM. Blood concentrations of nonesterified fatty acids before calving and β-hydroxy-butyrate after calving were not affected by treatment. Supplementation with the DFM product increased milk fat percentage for the first lactation cows and increased milk protein percentage for the second and greater lactation cows during the first 85 DIM. Second-lactation cows fed the DFM product received fewer antibiotic treatments before 85 DIM than cows receiving the placebo. This validated the dairy producer's concern that cows consuming one of the diets (revealed to be the placebo diet after the study was completed) were experiencing more health problems. Most measures of milk yield were numerically increased by supplementation with the DFM product. However, differences in milk yield were not significant. Key covariates for main study outcomes included milk yield in the previous (first) lactation, body condition score prior to calving, days spent in the maternity pen, and stocking density of the pre-fresh pen.     

Effects of supplemental dietary biotin on performance of Holstein cows during early lactation

This study determined the effects of supplemental dietary biotin (0, 10, or 20 mg/d) on performance of Holstein cows (n = 45; 18 primiparous and 27 multiparous). Treatments started at 14 d prepartum and continued until 100 d in milk (DIM). Blood samples were taken at 14 d prepartum, and blood and milk samples were taken at calving, and 30, 60, and 100 DIM. Dry matter intake during lactation was not different across treatments (19.7 kg/d). Milk production linearly increased with biotin supplementation (36.9, 37.8, and 39.7 kg/d for 0, 10, and 20 mg/d of supplemental biotin, respectively). Biotin supplementation did not affect milk fat and true protein percentages or fat yield but linearly increased true protein yield. Supplemental biotin increased concentrations of biotin in plasma and milk at all time points. Concentrations of biotin in plasma and milk (colostrum) at calving were higher than at other time points for cows fed supplemental biotin. In an ancillary experiment, plasma biotin concentrations were not as high when cows were fed 20 mg/d of supplemental biotin for 14 d during the middle of their dry period as when cows were fed 20 mg/d of biotin for the last 14 d of gestation. This suggests that events associated with parturition altered plasma biotin concentrations. Plasma concentrations of glucose, insulin, nonesterified fatty acids, and molar proportions of ruminal volatile fatty acids were not affected by biotin supplementation. Biotin supplementation had no effect on change in body weight or condition score. Supplemental biotin linearly increased milk and protein yields, however, the mode of action that caused these increases was not determined.     

Effect of bovine somatotropin (500 mg) administered at ten-day intervals on ovulatory responses, expression of estrus, and fertility in dairy cows

The objectives of this study were to evaluate the effect of administering 500 mg of recombinant bovine somatotropin (bST) every 10 d on ovulatory responses, estrous behavior, and fertility of lactating Holstein cows. Lactating dairy cows were assigned to 1 of 2 treatments: a control with no administration of bST (73 primiparous and 120 multiparous cows) or 6 consecutive administrations of 500 mg of bST (83 primiparous and 123 multiparous cows) given subcutaneously at 10-d intervals starting 61 ± 3 d postpartum (study d 0), concurrent with the initiation of the timed artificial insemination (AI). Blood samples were collected thrice weekly from 61 ± 3 to 124 ± 3 d in milk (DIM), and plasma samples were analyzed for concentrations of estradiol, glucose, insulin, insulin-like growth factor 1, and progesterone. The estrous cycle of cows was presynchronized with 2 injections of PGF_2α at 37 ± 3 and 51 ± 3 DIM, and the Ovsynch timed AI protocol was initiated at 61 ± 3 DIM. Ovaries were scanned to determine ovulatory responses during the Ovsynch protocol. Pregnancy was diagnosed at 33 and 66 d after AI. Body condition was scored on study d 0, 10, 42, and 76. Sixty-four cows were fitted with a pressure mounting sensor with radiotelemetric transmitters to monitor estrous behavior. Treatment of lactating dairy cows with 500 mg of bST at 10-d intervals increased yields of milk and milk components in the first 2 mo after treatment. Body condition of bST-treated cows remained unaltered, whereas control cows gained BCS. Treatment with bST increased concentrations of insulin-like growth factor 1 chronically, but concentrations of insulin and glucose increased only transiently in the first 7 d after the first injection of bST. Concentrations of progesterone during and after the Ovsynch protocol remained unaltered after treatment with bST; likewise, ovulatory responses during the Ovsynch protocol were mostly unaltered by treatment. Concentration of estradiol tended to be greater for bST cows than for control cows immediately before induction of ovulation in the Ovsynch protocol. Similarly, the mean and the peak concentrations of estradiol were greater for bST cows than for control cows when monitored during spontaneous estrus. Nevertheless, duration of estrus and the median number of standing events were less for bST cows than for control cows. Pregnancies per AI after the first and second postpartum inseminations were not affected by bST treatment. Treatment of lactating dairy cows with 500 mg of bST every 10 d improved lactation performance, but it did not affect pregnancies per AI and it reduced expression of estrus.     

Natural variation in biomarkers indicating mastitis in healthy cows

Dairy herds are expanding and, with increasing numbers of animals in each herd, there is a need for automatic recording of indicators in milk in order to detect mastitis, inflammation of the udder. A number of biomarkers for mastitis have been suggested over the years. Mastitis usually occurs in one of the four udder quarters and since it is now possible to milk each udder quarter separately in automated milking systems, it is important to evaluate the normal variation in the biomarkers at udder quarter level. This study evaluated the normal variations between milkings for some biomarkers in clinically healthy cows, determined by repeated somatic cell count and bacteriological analysis. The biomarkers studied were serum amyloid A (SAA), haptoglobin (Hp), lactate dehydrogenase (LDH), N-acetyl-β-D-glucosaminidase (NAGase) and alkaline phosphatase (AP), parameters that have been suggested as markers for mastitis. Ten cows were monitored on 42 consecutive milking occasions through collection of udder quarter milk samples and representative cow composite milk samples, giving a total of 2100 individual milk samples. Each cow had its individual profile for the concentrations and variations in the parameters analysed. Although there was relatively large variation between cows for the biomarkers analysed, the variation between milkings in clinically healthy quarters within cows was often below 10%. The biomarker with the lowest variation in this study was LDH. The results suggest that comparing quarters within an individual cow can identify deviations from the natural variations between milkings. This could be a valuable tool instead of, or in combination with, a cut-off value for each parameter in order to detect changes in the milk indicating mastitis.