Estrone and 17β-estradiol concentrations in pasteurized-homogenized milk and commercial dairy products

Some individuals fear that estrogens in dairy products may stimulate growth of estrogen-sensitive cancers in humans. The presence of estrone (E₁) and 17β-estradiol (E₂) in raw whole cow's milk has been demonstrated. The objectives of this study were to determine if pasteurization-homogenization affects E₂ concentration in milk and to quantify E₁ and E₂ concentrations in commercially available dairy products. The effects of pasteurization-homogenization were tested by collecting fresh raw milk, followed by pasteurization and homogenization at 1 of 2 homogenization pressures. All treated milks were tested for milk fat globule size, percentages of milk fat and solids, and E₂ concentrations. Estrone and E₂ were quantified from organic or conventional skim, 1%, 2%, and whole milks, as well as half-and-half, cream, and butter samples. Estrone and E₂ were quantified by RIA after organic solvent extractions and chromatography. Pasteurization-homogenization reduced fat globule size, but did not significantly affect E₂, milk fat, or milk solids concentrations. Estrone concentrations averaged 2.9, 4.2, 5.7, 7.9, 20.4, 54.1 pg/mL, and 118.9 pg/g in skim, 1%, 2%, and whole milks, half-and-half, cream, and butter samples, respectively. 17β-Estradiol concentrations averaged 0.4, 0.6, 0.9, 1.1, 1.9, 6.0 pg/mL, and 15.8 pg/g in skim, 1%, 2%, whole milks, half-and-half, cream, and butter samples, respectively. The amount of fat in milk significantly affected E₁ and E₂ concentrations in milk. Organic and conventional dairy products did not have substantially different concentrations of E₁ and E₂. Compared with information cited in the literature, concentrations of E₁ and E₂ in bovine milk are small relative to endogenous production rates of E₁ and E₂ in humans.     

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.     

Plasma progesterone concentration after first service is associated with individual genetic traits,postpartum phenotypes,and likelihood of conception in seasonal-calving pasture-based dairy cows

The aims of this study were to (1) evaluate postpartum phenotypes, cow factors, and genetic traits associated with plasma progesterone (P4) concentrations after first artificial insemination (AI); (2) determine variation in daily plasma P4 concentrations between d 7 and 13 after first AI; and (3) evaluate associations between plasma P4 concentrations and pregnancy success after first AI. First and second parity (n = 2,797) spring-calving lactating dairy cows from 35 dairy herds were enrolled. Farm visits were performed every 2 wk during the postpartum period as follows: cows that were at wk 3 (range: 14–27 d in milk) and wk 7 (range: 42–55 d in milk) postpartum were examined. Farm visits were performed weekly during the breeding season, and cows that were between 7 and 13 d after the first AI were examined. Body condition score (BCS) was measured at each visit using a 1 to 5 scale [low (≤2.75), target (≥3.0)]. Transrectal ultrasound examinations were conducted at wk 3 and wk 7 postpartum visits to determine presence or absence of a corpus luteum (CL) and uterine tract score [scale of G₁ (best)–G₄ (worst)]. Blood samples were collected at each visit, and plasma concentrations of glucose, β-hydroxybutyrate, and fatty acids were analyzed. On the day of the weekly farm visit during the breeding season, blood samples for P4 determination were collected from all cows that were between 7 and 13 d after first AI during the breeding period. Cows that had a CL present and a G₁ uterine score at wk 7 postpartum had greater plasma P4 concentration after first AI (+0.67 ng/mL and +0.4 ng/mL, respectively) compared with cows with no CL present and with a uterine score ≥G₃. Cows with low BCS at wk 7 postpartum had lesser plasma P4 concentration after first AI than cows with target BCS. Each unit increase in plasma fatty acids and β-hydroxybutyrate concentration at AI was associated with 0.45 ± 0.33 ng/mL (estimate ± standard error) and 0.07 ± 0.04 ng/mL greater plasma P4 concentration after first AI, respectively. Regarding genetic merit traits, each unit increase in fertility subindex was associated with 0.005 ± 0.003 ng/mL greater P4 concentration. In addition, for every 1 ng/mL increase in plasma P4 concentration, the odds of estimated probability of pregnancy per AI increased by 3% (odds ratio = 1.03; 95% confidence interval = 1.00, 1.05). In conclusion, cows with superior genetic merit for fertility traits and milk production traits, favorable fertility phenotypes at wk 7 postpartum, (e.g., presence of a CL, a G₁ uterine score, and target BCS), and blood parameters indicative of better metabolic status at AI were all associated with greater plasma P4 concentration after AI. In turn, greater plasma P4 concentrations were associated with greater odds of successful pregnancy establishment. This study underlines the important associations between early postpartum fertility phenotypes (CL presence, uterine health status) and subsequent plasma P4 concentrations after first AI, and hence provides additional evidence of the mechanisms through which selection for fertility traits improves phenotypic fertility performance.     

Short communication: Relationships among plasma and milk vitamin B12, plasma free fatty acids,and blood β-hydroxybutyrate concentrations in early lactation dairy cows

This study was undertaken to evaluate the relationship between plasma and milk concentrations of vitamin B₁₂ as well as the relationship between plasma or milk concentrations of vitamin B₁₂ and plasma concentration of free fatty acids (FFA) or blood concentration of β-hydroxybutyrate (BHB) of early lactating Ayrshire (AY) and Holstein (HO) cows. A total of 44 dairy herds (7 AY and 37 HO herds) and 62 AY (21 in first, 19 in second, and 22 in third and more lactations) and 228 HO (51 in first, 74 in second, and 103 in third and more lactation) cows between 3 and 40 d in milk were involved in the study. Hand-stripped milk samples and blood samples were taken 6 h after the morning milking. Milk and plasma samples were analyzed for vitamin B₁₂ concentration and plasma samples were analyzed for FFA concentration. A handheld device was used for blood BHB concentration determination. Thresholds for elevated plasma FFA concentration and hyperketonemia were set at ≥0.70 and ≥1.2 mmol/L, respectively. Vitamin B₁₂ concentration in milk of AY primiparous cows [2,557 (1,995–3,276) pg/mL] was lower than in milk from HO primiparous cows [3,876 (3,356–4,478) pg/mL], whereas no difference was observed among other parities and breeds. Regardless of breeds, plasma concentration of vitamin B₁₂ of first and second parities was lower than plasma concentration of third and more lactation cows. Milk vitamin B₁₂ concentration was positively correlated with plasma vitamin B₁₂ concentration, but the relationship was stronger for AY (ρ averaging 0.63) than for HO cows (ρ averaging 0.36). For AY and HO breeds, a significant relationship between milk or plasma vitamin B₁₂ concentrations and plasma FFA concentration (ρ between 0.29 and 0.59) was observed. Moreover, cows with elevated plasma FFA concentration had greater milk and plasma vitamin B₁₂ concentrations than cows with normal plasma FFA concentration. No relationship between vitamin B₁₂ concentration in milk or plasma and blood BHB concentration and hyperketonemia was noted. In summary, milk is not well correlated with plasma vitamin B₁₂ concentration for HO. It could be hypothesized that elevated plasma concentration of FFA could have a negative effect on the use of vitamin B₁₂ by cow cells, which increases the concentration of the vitamin in plasma and its secretion in milk.     

Anti-Müllerian hormone in grazing dairy cows: Identification of factors affecting plasma concentration,relationship with phenotypic fertility,and genome-wide associations

The objectives of this study were to (1) characterize the distribution and variability of plasma anti-Müllerian hormone (AMH) concentration; (2) evaluate factors associated with phenotypic variation in plasma AMH; (3) examine the associations between categories of plasma AMH and reproductive outcomes [pregnancy to first artificial insemination (P/AI), and pregnancy rates within 21, 42, and 84 d after the mating start date (MSD)]; (4) estimate pedigree and genomic heritability for plasma AMH; and (5) identify and validate SNP associated with phenotypic variation in plasma AMH. Plasma AMH concentration (pg/mL) was determined from a blood sample collected (mean ± standard deviation) 10 ± 2 d after first insemination at detected estrus (IDE) in 2,628 first- and second-parity Irish dairy cows. Overall, plasma AMH had a positively skewed distribution with mean (± standard deviation), median, minimum, and maximum concentrations of 326 ± 231, 268, 15, and 2,863 pg/mL, respectively. Plasma AMH was greatest for Jersey, followed by Holstein × Jersey, Holstein × Norwegian Red, and Holstein cows (410, 332, 284, and 257 pg/mL, respectively). Second-parity cows had greater plasma AMH than first-parity cows (333 vs. 301 pg/mL, respectively). Samples collected at 7 and 8 d after first IDE had lesser plasma AMH than those collected on d 9, 10, 11, 12, and 13 after first IDE (291 and 297 vs. 317, 319, 331, 337, and 320 pg/mL). Plasma AMH was not associated with either body condition score at first IDE or the interval from calving to MSD. Cows were categorized into low (≤150 pg/mL; n = 526; lowest 20%), intermediate (>150 to ≤461 pg/mL; n = 1,576; intermediate 60%), and high AMH (>461 pg/mL; n = 526; highest 20%) groups based on plasma AMH, and associations with reproductive outcomes were tested. Cows with high and intermediate plasma AMH had 1.42- and 1.51-times-greater odds of becoming pregnant within 84 d after the MSD than those with low plasma AMH (90.3 and 90.8 vs. 86.8%, respectively); however, P/AI and pregnancy rate within 21 and 42 d after the MSD did not differ among AMH categories. Plasma AMH was moderately heritable (pedigree heritability of 0.40 ± 0.06 and genomic heritability of 0.45 ± 0.05), and 68 SNP across Bos taurus autosomes 7 and 11 were associated with phenotypic variation in plasma AMH. Out of 68 SNP, 42 were located in a single quantitative trait locus on Bos taurus autosome 11 that harbored 6 previously identified candidate genes (NR5A1, HSPA5, CRB2, DENND1A, NDUFA8, and PTGS) linked to fertility-related phenotypes in dairy cows.     

Short communication: Heat treatment and souring do not affect milk estrone and 17β-estradiol concentrations

The aim of our study was to establish whether heat treatment and souring of milk affect its estrone (E1) and 17β-estradiol (E2) concentrations. Milk samples were collected from 10 Holstein cows in late pregnancy. Concentrations of E1 and E2 were measured in milk samples that were previously heated to 70 and 95°C for 5 min. Additionally, E1 and E2 concentrations were determined in the same milk samples after 2 d of spontaneous souring at room temperature, and these samples were compared with E1 and E2 levels in raw, unprocessed milk. Concentrations of both hormones were determined by commercial ELISA kits. Concentrations of E1 in unprocessed and processed milk (milk heated to 70 and 95°C and soured milk) were (mean ± SE) 47.25 ± 4.16, 44.84 ± 3.47, 41.00 ± 4.55, and 44.92 ± 3.91 pg/mL, respectively. Concentrations of E2 in the same milk samples were 36.11 ± 10.01, 32.46 ± 9.88, 31.78 ± 9.56, and 31.43 ± 8.00 pg/mL, respectively. Concentrations of E1 and E2 in heat-treated milk did not differ significantly from those in unprocessed milk. Similarly, E1 and E2 concentrations in soured milk did not differ significantly from those in unprocessed milk samples. These results indicate that E1 and E2 are stable in milk and that milk processing (heating and souring) does not influence their degradation. Therefore, E1 and E2 concentrations are expected to be similar between commercial full-fat milk and the raw milk from which it was produced.     

Milk and serum IL-4, IL-6, IL-10, and amyloid A concentrations in cows with subclinical mastitis caused by coagulase-negative staphylococci

The aim of the study was to evaluate the concentrations of cytokines IL-4, IL-6, and IL-10 and acute phase protein amyloid A in milk and in serum from cows with subclinical mastitis caused by coagulase-negative staphylococci and from healthy cows. The blood and milk samples were obtained from 35 midlactation, multiparous (between parities 2 and 4) Holstein-Friesian cows. In the milk samples from 20 cows with subclinical mastitis, the following species of Staphylococcus were detected: Staphylococcus xylosus (8 samples), Staphylococcus chromogenes (6 samples), Staphylococcus haemolyticus (2 samples), Staphylococcus simulans (2 samples), and Staphylococcus sciuri (2 samples). The results of the present study indicate that the level of IL-6 in cows suffering from subclinical mastitis tended to be high in both serum and milk (432.09 and 254.32 pg/mL) compared with the level in healthy cows (164.47 and 13.02 pg/mL, respectively). Amyloid A value also was significantly higher in milk of unhealthy cows compared with cows without subclinical mastitis (790.2 and 360.5 ng/mL). No significant differences were found in levels of amyloid A in serum of both tested groups of cows (2,680.0 and 2,720.0 ng/mL). In contrast, concentration of IL-4 was significantly lower both in serum and in milk of cows with staphylococcal mastitis (86.1 and 123.17 pg/mL) compared with control animals (413.5 and 670.2 pg/mL). The level of IL-10 also was significantly higher in milk of healthy cows than in infected cows (39.78 and 22.5 pg/mL); however, differences in serum levels of this cytokine between tested groups were significantly less important (220.6 and 175.1 pg/mL).     

The relationship between serum anti-Müllerian hormone concentrations and fertility,and genome-wide associations for anti-Müllerian hormone in Holstein cows

The objectives of this study were to (1) evaluate factors associated with variation in circulating anti-Müllerian hormone (AMH) concentrations, (2) establish an optimum AMH threshold predictive of pregnancy to first artificial insemination (P/AI), (3) examine the relationship between AMH and fertility (P/AI, pregnancy loss between 30 and 60 d after artificial insemination, and pregnancy risk up to 250 d postpartum), and (4) identify quantitative trait loci associated with phenotypic variation of AMH concentrations in dairy cows. Serum AMH concentrations (pg/mL) were determined at 7 ± 2.4 d postpartum in 647 lactating Holstein cows (213 primiparous, 434 multiparous) from 1 research and 6 commercial dairy herds in Alberta, Canada. Of these, 589 cows were genotyped on the 26K Bovine BeadChip (Neogen Inc., Lincoln, NE) and subsequently imputed to the Illumina Bovine High Density BeadChip (Illumina, San Diego, CA) for genome-wide association analysis for variation in serum AMH concentrations. Factors associated with variation in serum AMH concentrations and the relationship between categories of AMH and aforementioned fertility outcomes were evaluated only in a subset of 460 cows that had a complete data set available. The overall mean (±standard error of the mean), median, minimum, and maximum AMH concentrations were 191.1 ± 6.3, 151.7, 13.9, and 1,879.0 pg/mL, respectively. The AMH concentrations were not associated with herd, precalving body condition score, postpartum week, and season of sampling; the lactation number, however, had a quadratic relationship with serum AMH concentrations (116.2, 204.9 204.5, and 157.9 pg/mL for first, second, third, and ≥fourth lactation, respectively). The optimum AMH threshold predictive of P/AI could not be established because the receiver operating characteristic curve analysis model was nonsignificant. Categories of AMH [low (<83.0 pg/mL; n = 92), intermediate (≥83.0 to ≤285.0 pg/mL; n = 276), and high (>285.0 pg/mL; n = 92) based on lowest 20%, intermediate 60%, and highest 20% serum AMH) had no associations with P/AI (34, 43, and 40%), pregnancy loss between 30 and 60 d after artificial insemination (20, 12, and 8%), or pregnancy risk up to 250 d postpartum. One candidate gene associated with AMH production [AMH gene on Bos taurus autosome (BTA) 7] and 4 candidate genes related to embryo development (SCAI and PPP6C genes on BTA11 and FGF18 and EEF2K genes on BTA20 and BTA25, respectively) were in linkage disequilibrium with single nucleotide polymorphisms associated with phenotypic variation in serum AMH in dairy cows.     

Effects of clay after an aflatoxin challenge on aflatoxin clearance,milk production,and metabolism of Holstein cows

Oral supplementation of clay to dairy cattle has been reported to reduce toxicity of aflatoxin (AF) in contaminated feed. The objective of this study was to determine the effects of 3 concentrations of dietary clay supplementation in response to an AF challenge. Ten multiparous rumen-cannulated Holstein cows [body weight (mean ± SD) = 669 ± 20 kg and 146 ± 69 d in milk], were assigned to 1 of 5 treatments in a randomized replicated 5 × 5 Latin square design balanced to measure carryover effects. Periods (21 d) were divided in an adaptation phase (d 1 to 14) and a measurement phase (d 15 to 21). From d 15 to 17, cows received an AF challenge. The challenge consisted of 100 μg of aflatoxin B₁ (AFB₁)/kg of dietary dry matter intake (DMI). The material was fitted into 10-mL gelatin capsules and administered into the rumen through a rumen-cannula based on the average DMI obtained on d 12 to 14. Treatments were no clay plus an AF challenge (POS); 3 different concentrations of clay (0.5, 1, or 2% of dietary DMI) plus an AF challenge; and a control consisting of no clay and no AF challenge (C). Statistical analysis was performed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). Two contrasts, CONT1 (POS vs. C) and CONT2 (POS vs. the average of 0.5, 1, and 2% clay), were compared along with the linear and quadratic treatment effects (POS, 0.5%, 1%, 2%). Cows supplemented with clay had lower AF excretion in milk as aflatoxin M₁ (AFM₁; 0.5% = 20.83 μg/d, 1% = 22.82 μg/d, and 2% = 16.51 μg/d) and AF transfer from rumen fluid to milk (AFM_1; 0.5% = 1.01%, 1% = 0.98%, and 2% = 0.74%) compared with cows in POS (AFM₁ = 27.81 μg/d and AF transfer = 1.37%, CONT2). Similarly, concentrations of AFM₁ in milk (0.5% = 0.35 μg/kg, 1% = 0.30 μg/kg, 2% = 0.25 μg/kg), AFB₁ in feces (0.5% = 1.79 μg/g, 1% = 1.52 μg/kg, 2% = 1.48 μg/kg), and AFB₁ in rumen fluid (0.5% = 0.05 μg/kg, 1% = 0.02 μg/kg, 2% = 0.02 μg/kg) were reduced in cows fed clay compared with POS (0.43 μg/kg, 2.78 μg/kg, and 0.10 μg/kg, respectively, CONT2). Cows supplemented with clay tended to have lower 3.5% fat-corrected milk [0.5% = 38.2 kg, 1% = 39.3 kg, 2% = 38.4 kg, standard error of the mean (SEM) = 1.8] than cows in POS (41.3 kg; SEM = 1.8; CONT2). Plasma superoxide dismutase (SOD) concentration tended to be lower for cows fed clay in the diet (0.5% = 2.16 U/mL, 1% = 1.90 U/mL, 2% = 2.3 U/mL; SEM = 0.3) than for cows in POS (2.72 U/mL; CONT2). Additionally, when cows were exposed to AF without clay in the diet, plasma concentrations of aspartate aminotransferase (AST) decreased from 84.23 (C) to 79.17 (POS) and glutamate dehydrogenase (GLDH) decreased from 91.02 (C) to 75.81 (POS). In conclusion, oral supplementation of clay reduced the transfer of AF from the rumen to milk and feces.     

Increased dietary vitamin D3 and calcium partially alleviate heat stress symptoms and inflammation in lactating Holstein cows independent of dietary concentrations of vitamin E and selenium

Twelve multiparous Holstein cows (42.2 ± 5.6 kg of milk/d; 83 ± 27 d in milk) were used in a split-plot design testing the effects of mineral and vitamin supplementation on the time course of animal performance, metabolism, and inflammation markers during heat stress. The main plot was the average concentrations of dietary vitamin E and Se (adequate: 11.1 IU/kg of vitamin E and 0.55 mg/kg of Se, and high: 223 IU/kg of vitamin E and 1.8 mg/kg of Se, respectively). Within each plot, cows were randomly assigned to (1) heat stress (HS) with adequate concentrations of vitamin D₃ and Ca (1,012 IU/kg and 0.73%, respectively), (2) HS with high concentrations of vitamin D₃ and Ca (HS+D₃/Ca; 3,764 IU/kg and 0.97%, respectively), or (3) pair-feeding (PF) in thermoneutrality with adequate concentrations of vitamin D₃ and Ca (1,012 IU/kg and 0.73% Ca) in a Latin square design with 14-d periods and 7-d washouts. The highest rectal temperature was recorded at 1700 h for HS (39.4°C; mean of d 1 to 14), being 1.2 and 0.8°C greater than for PF and HS+D₃/Ca, respectively. Respiratory rate and water intake were higher in HS (73 breaths/min and 115 L/d, respectively) relative to PF (28 breaths/min and 76 L/d). Heat stress decreased dry matter intake progressively, reaching a nadir on d 5 to 7 (33% reduction) and was not different between treatments. Milk yield decreased progressively in all treatments, but remained greater in PF relative to HS from d 3 to 14 (10%), whereas HS and HS+D₃/Ca were not different. Milk fat, protein, and lactose concentrations and yields were lower in HS relative to PF from d 3 to 14, but not different between HS and HS+D₃/Ca. Relative to PF, preprandial insulin concentrations were increased in HS, whereas plasma nonesterified fatty acids were decreased on d 7 and 14. Plasma lipopolysaccharide-binding protein concentrations increased in HS cows on d 7 and 14, respectively, relative to PF, whereas they were reduced in HS + D₃/Ca on d 14. Plasma C-reactive protein, tumor necrosis factor-α, and fecal calprotectin were increased in HS relative to both PF and HS+D₃/Ca on d 7 and 14. Rectal temperature was positively associated with plasma lipopolysaccharide-binding protein (r = 0.72), tumor necrosis factor-α (r = 0.74), C-reactive protein (r = 0.87), and with milk somatic cells (r = 0.75). Plasma 8-hydroxy-2-deoxyguanosine concentrations presented a 3-way interaction, where 8-hydroxy-2-deoxyguanosine was lower in HS than in PF on d 7 and 14, and lower in HS+D₃/Ca relative to HS on d 14 in the adequate vitamin E and Se treatment, but no effects were observed in the high vitamin E and Se group. Plasma superoxide dismutase concentrations increased over time, and were higher in HS relative to PF on d 14, whereas HS+D₃/Ca was similar to HS. Heat stress markedly reduced milk production and milk components while increasing markers of leaky gut and inflammation. In contrast, vitamin D₃ and Ca supplementation reduced hyperthermia (d 7–14), markers of leaky gut, and inflammation independent of dietary concentrations of vitamin E and Se.