Prolonged exposure to high environmental temperatures results in an accumulated heat load that induces a heat stress (HS) response in dairy cattle. Heat stress compromises dairy farm profitability by reducing milk yield, altering milk composition, and hindering reproductive performance. The ability to alternate between carbohydrate and lipid sources for energy production is termed metabolic flexibility (Met Flex). The objective of this study was to evaluate the Met Flex of mammary, muscle, and liver tissue in lactating dairy cows under HS and thermoneutral (TN) conditions. Sixteen Holstein cows were assigned to 1 of 2 treatment groups: pair-feeding in TN conditions (PFTN) or HS conditions. All cows experienced a 4-d TN period with ad libitum intake followed by a 4-d treatment period. Heat stress cows were exposed to a temperature-humidity index (THI) ranging from 76 to 80 and the PFTN cows were exposed to a THI of 64. Milk production and health data were recorded twice daily. Semitendinosus biopsies were obtained on d 4 of each period and postmortem mammary and liver samples were obtained on d 4 of period 2. All tissue samples were assayed for Met Flex. Activity of mitochondrial (Mit) enzymes were assessed in skeletal muscle only. Four days of HS decreased milk yield, altered milk composition, and increased respiration rate and rectal temperatures. No differences in Met Flex were observed in mammary or liver tissue during period 2. However, HS, but not PFTN conditions, lowered Met Flex of skeletal muscle by 18.3% when compared with TN ad libitum feed intake conditions of period 1. No treatment differences were observed in skeletal muscle Mit enzyme activity indicating the decrease in Met Flex occurred independently of changes in Mit function. The reduction in Met Flex of skeletal muscle during HS may contribute to reduced milk yield and warrants further investigation.

Maternal supplementation with omega-3 fatty acids during late gestation has been shown to have a variety of benefits for neonates. The current study utilized 13 mares and their foals, assigned to one of three dietary treatments: an unsupplemented control diet (CON; n = 5), or the addition of either fish oil-derived omega-3 (FO; n = 5) or a flaxseed (FLAX; n = 3) supplement. The mares received treatments beginning on day 310 of gestation through day 5 post-parturition. Samples of plasma were collected from the mares prior to beginning supplementation, on days 0, 5, and 30 post-partum. Milk samples were collected from the mares 12 h post-partum. Plasma samples were collected from the foals at birth and at 5 and 30 d post-partum. The fatty acid compositions of the mare plasma, mare milk, and foal plasma were determined using gas chromatography. The data were analyzed using repeated-measures ANOVA. Foals born to FO mares exhibited increased plasma docosahexaenoic acid (DHA) levels at birth compared with those born to CON mares. Across the treatments, the plasma from foals at birth prior to nursing had the highest DHA content compared with on d 5 and 30. No treatment differences were observed in the mare plasma or milk fatty acid composition for DHA levels. FLAX mares had higher 20:3 n6 plasma proportions than FO or CON mares. Beginning supplementation with fish oil on day 310 of gestation may be sufficient to elevate neonatal foal DHA concentrations.

Heat stress (HS) is a global issue that decreases farm profits and compromises animal welfare. To distinguish between the direct and indirect effects of HS, 16 multiparous Holstein cows approximately 100 DIM were assigned to one of 2 treatments: pair fed to match HS cow intake, housed in thermoneutral conditions (PFTN, n = 8) or cyclical HS (n = 8). All cows were subjected to 2 experimental periods. Period 1 consisted of a 4 d thermoneutral period with ad libitum intake. During period 2 (P2), the HS cows were housed in cyclical HS conditions with a temperature -humidity index (THI) ranging from 76 to 80 and the PFTN cows were exposed to a constant THI of 64 for 4 d. Dry matter intake of the PFTN cows was intake matched to the HS cows. Milk yield, milk composition, rectal temperature, and respiration rate were recorded twice daily, blood was collected daily via a jugular catheter, and cows were fed twice daily. On d 3 of each period, Cr-EDTA and sucralose were orally administered and recovered via 24 h total urine collection to assess gastrointestinal permeability. All data were analyzed using the GLIMMIX procedure in SAS. The daily data collected in P1 was averaged and used as a covariate if deemed significant in the model. Heat stress decreased voluntary feed intake by 35% and increased rectal temperature and respiration rate (38.4 degrees C vs. 39.4 degrees C and 40 vs. 71 respirations/min, respectively). Heat stress reduced DMI by 35%, which accounted for 66% of the decrease in milk yield. The yields, and not concentrations, of milk protein, fat, and other solids were lower in the HS cows on d 4 of P2. Milk urea nitrogen was higher and plasma urea nitrogen tended to be higher on d 3 and d 4 of HS. Glucose was 7% lower in the HS cows and insulin was 71% higher in the HS cows than the PFTN cows on d 4 of P2. No difference in lipopolysaccharide-binding protein was observed. Heat stress cows produced 7 L/d more urine than PFTN cows. No differences were detected in the urine concentration or percentage of the oral dose recovered for Cr-EDTA or sucralose. In conclusion, HS was responsible for 34% of the reduction of milk yield. The elevated MUN and the tendency for elevated plasma urea nitrogen indicate a whole-body shift in nitrogen metabolism. No differences in gastrointestinal permeability or lipopolysaccharide-binding protein were observed. These results indicate that, under the conditions of this experiment, activation of the immune system by gutderived lipopolysaccharide was not responsible for the decreased milk yield observed during HS.

Milk yield (MY) naturally declines after peak lactation. One approach to improving production is to increase lactation persistency, or the length of time a cow maintains lactation. To do so, cows must maintain functional secretory mammary epithelial cells (MEC), which synthesize milk. Some viable MEC are exfoliated from the udder into milk. The objective of this experiment was to identify the magnitude and direction of association between exfoliated MEC and MY, days in milk (DIM), and parity. Our hypothesis was that MY and the quantity of exfoliated MEC were negatively correlated. Therefore, milk from low yielding, late lactation, and primiparous cows would contain more MEC. Thirty-six multiparous and 12 primiparous Holstein cows were sampled once during lactation. A single milk sample was obtained from each cow during a morning milking. Flow cytometry was used to identify milk cell types. Cells were stained with an antibody specific for cluster of differenciation-45 (CD45) to label hematopoietic cells and exclude them from analysis. An antibody against butyrophilin 1A1 (BTN), a protein involved in milk fat secretion, labeled MEC. Nucleated cells were stained with Hoechst dye, and propidium iodide was used to identify dead cells. For statistical analysis, we analyzed the effect of parity, MY, and DIM, and DIM2 on the number and concentration of both total and live exfoliated MEC. Primiparous cows exfoliated fewer MEC and had a lower concentration of MEC in milk than multiparous cows regardless of lactation stage. This could reflect continued mammary growth in first lactation. Cows shed an average of 9.80x107 ± 6.907 MEC with a concentration of 4,582 ± 3,574 cells/mL in one milking. Milk yield was inversely related to concentration of both total and live MEC in milk. Similarly, cows in late lactation, which is associated with MY decline, exfoliated a greater number of MEC into milk. Primiparous cows had a lower milk yield and shed fewer total and live MEC in this study. The relationship of MY and DIM with MEC losses in milk may also contribute to declining MEC number in the mammary gland found in post-peak lactation. Understanding MEC exfoliation regulation could help develop husbandry practices that improve secretory cell retention in the mammary gland and increase lactation persistency.

This study aimed to evaluate the impact of heat stress on mammary epithelial cell (MEC) losses into milk, secretory mammary tissue structure, and mammary epithelial cell activity. Sixteen multiparous Holstein cows (632 ± 12 kg BW) approximately 100 d in milk housed in climate-controlled rooms were paired by body weight and randomly allocated to one of 2 treatments, heat stress (HS) or pair feeding thermoneutral (PFTN) using 2 cohorts. Each cohort was subjected to 2 periods of 4 d each. In period 1, both treatments had ad libitum access to a common total mixed ration and were exposed to a controlled daily temperature-humidity index (THI) of 64. In period 2, HS cows were exposed to controlled cyclical heat stress (THI: 74 to 80), while PFTN cows remained at 64 THI and daily dry matter intake was matched to HS. Cows were milked twice daily, and milk yield was recorded at each milking. Individual milk samples on the last day of each period were used to quantify MEC losses by flow cytometry using butyrophilin as a cell surface marker. On the final day of period 2, individual bovine mammary tissue samples were obtained for histomorphology analysis, assessment of protein abundance, and evaluation of gene expression of targets associated with cellular capacity for milk and milk component synthesis, heat response, cellular proliferation, and autophagy. Statistical analysis was performed using the GLIMMIX procedure of SAS. Milk yield was reduced by 4.3 kg by HS (n = 7) compared with PFTN (n = 8). Independent of treatment, MEC in milk averaged 174 cells/mL (2.9% of total cells). There was no difference between HS vs. PFTN cows for MEC shed or concentration in milk. Alveolar area was reduced 25% by HS, and HS had 4.1 more alveoli than PFTN. Total number of nucleated MEC per area were greater in HS (389 ± 1.05) compared with PFTN (321 ± 1.05); however, cell number per alveolus was similar between groups (25 ± 1.5 vs. 26 ± 1.4). There were no differences in relative fold expression for GLUT1, GLUT8, CSN2, CSN3, LALBA, FASN, HSPA5, and HSPA8 in HS compared with PFTN. Immunoblotting analyses showed a decrease abundance for phosphorylated STAT5 and S6K1, and an increase in LC3 II in HS compared with PFTN. These results suggest that even if milk yield differences and histological changes occur in the bovine mammary gland after 4 d of heat exposure, MEC loss into milk, nucleated MEC number per alveolus, and gene expression of nutrient transport, milk component synthesis, and heat stress related targets are unaffected. In contrast, the abundance of proteins related to protein synthesis and cell survival decreased significantly, while an upregulation of proteins associated with autophagy in HS compared with PFTN.

Somatic cells (SCs) in milk are a heterogeneous population composed of several subsets of cells. However, a complete understanding of this heterogeneity in cow’s milk remains elusive. This study aimed to characterize heterogeneity within mammary epithelial (MEC) and immune cell subpopulations from healthy Holstein cows. An initial cell characterization of SC populations was completed using a single milk collection (3.8 L) from a base population of 25 multiparous Holstein cows to identify MEC and immune cells using flow cytometry with Butyrophilin 1A1 (BTN) and CD45 as cell surface markers. From the base population, 5 multiparous cows (≥300 days in milk (DIM), ≤162 × 103 SC/mL, and milk yield (MY) ≥ 25 kg/d) were selected for fluorescence activated cell sorting and single-cell RNA sequencing (scRNA-seq) analysis. A single-cell-suspension of approximately 1,000 sorted cells was prepared from each cow for characterization using scRNA-seq. Gel beads and barcodes were generated, cDNA amplified, cDNA sequencing libraries constructed and sequenced. After data normalization, scaling, and filtering control, two CD45+ databases were generated. The CD45+ databases contained 923 and 851 single cells, each comprising 17,771 and 12,156 features, respectively. Principal component analysis revealed seven and eight distinguishing clusters. Based on marker expression, most immune cells present in the samples were T cells (CD3E and PTPRC). Three different T cell subpopulations were revealed: helpers (CD4), cytotoxic (CD8A and CD8B), and regulatory T cells (IL2RA). The remaining four clusters were composed of granulocytes (neutrophils, eosinophils, and basophils; TLR4 and CXCL8), macrophages (PTPRC, CD14, CD68, TL2, IL1B), and a small population of B cells (CD19, CD22, and MS4A1). The study and characterization of immune cell subpopulations present in milk provide a basis for developing greater insights into mammary gland immune function, offering potential avenues for enhancing animal health and milk production in the future.

This study aimed to evaluate the effect of feeding diets with different fractions of undegraded NDF (uNDF) and potentially degradable (pdNDF) on ruminal NDF degradation and passage kinetics of lactating dairy cows. Six rumen-cannulated (533 ± 43 kg BW and 122 ± 15 DIM) and 6 non-cannulated (558 ± 62 kg BW and 126 ± 16 DIM) primiparous Holstein dairy cows were randomly assigned to 1 of 2 experimental diets in a crossover design with 2 28-d periods. The experimental diets were formulated to include either alfalfa hay (ALFA) or orchardgrass hay (ORCH) in addition to corn silage. Rations were formulated to contain 30% NDF (DM basis), where the concentrate, corn silage, and each of the hays provided one third of the dietary NDF. The marker dilution technique was used to measure the passage rate utilizing a pulse dose of marked corn silage fiber. On d 17 and 24 of each period, ruminal contents were evacuated to determine ruminal pool size. Following the return of the ruminal contents containing the pulse dose of marked corn silage to the rumen, ruminal grab samples were collected at 0, 3, 6, 9, 12, 24, 36, 48, 60, and 72 h. Samples from each time point were separated into solids and liquid, and the solids were analyzed for NDF, uNDF, and marker concentration. Alfalfa hay had a higher concentration of CP (16.4 vs. 10.7%) and a lower concentration of NDF (38.0 vs. 63.2) than orchardgrass hay. Alfalfa hay had a greater concentration of uNDF than orchardgrass hay (36.5 vs. 32.8% uNDF; NDF basis). Cows consuming the ALFA diet had similar milk yield (39.1 kg/d) and similar milk fat and protein concentrations (3.72% fat and 3.24% protein, respectively) than cows consuming the ORCH diet. Cows consuming the ALFA diet consumed more DM (26.7 vs. 24.6 kg/d) and uNDF (2.7 vs. 2.3 kg/d), than cows consuming the ORCH diet. Cows consuming the ALFA diet digested more NDF and pdNDF than cows consuming the ORCH diet (3.3 vs. 2.8 kg/d). Even though cows consuming the ALFA diet had a smaller pool size of NDF than cows consuming the ORCH diet (5.4 vs. 6.7 kg), the pool size of uNDF did not differ between groups (2.4 kg). Cows consuming the ALFA diet had a faster rate of passage of uNDF than cows consuming the ORCH diet (5.02 vs. 4.03%/h). This translated into a shorter mean retention time of uNDF for cows consuming the ALFA diet relative to cows consuming the ORCH diet (21.0 vs. 26.2 h). In conclusion, cows consuming diets containing alfalfa hay had a faster ruminal passage rate and a shorter mean retention time of uNDF than cows consuming diets containing orchardgrass hay, and this occurred despite the greater concentrations of dietary uNDF in the alfalfa-based diet. These findings suggest that the kinetics of ruminal digestion and passage influence NDF degradation in ways beyond uNDF concentration or forage quality.

Dairy farm profitability depends on milk yield, so the dairy industry manages cows to improve their productivity. Both bovine somatotropin (bST) and early lactation increased milking frequency (IMF) and milk yield (MY) in dairy cows. The objective of this study was to evaluate the effects of mid-lactation bST administration on milk production in established lactation when combined with the milk yield carry-over effect from early lactation IMF. Thirteen multiparous Holstein cows were milked unilaterally for 20 days in early lactation. The left udder halves were milked twice daily (2X) and the right udder halves were milked four times daily (4X). Udder halves milked 4X produced 8.60 ± 1.40 kg more than 2X on the final day of IMF treatment. Cows were then returned to 2X milking for the remainder of lactation and sampled on alternate days from 74-94 days in milk (DIM). Bovine somatotropin was administered to all cows at 80 DIM. The 4X halves continued to make 2.66 ± 0.12 kg/d more milk than 2X through 94 DIM. Fat, protein, and lactose yields were significantly greater in the 4X halves compared to the 2X from 74-94 DIM. Overall milk yield increased by 2.71 kg/d with bST administration. However, there was no significant interaction between MF and bST administration. We can infer from these data that the mechanisms by which bST and IMF in early lactation increase milk yield are complementary due to their non-synergistic nature of enhancing MY.

Dairy farming requires forages with high neutral detergent fiber (NDF) to maximize milk production, sustain cows' health, and ensure the economic and environmental sustainability of the dairy farm. The objectives of this study were to determine the effects of the brown midrib (BMR) genotype, agronomic environment, and maturity at harvest on the NDF digestibility (NDFD) and the composition of the cell wall of corn plant tissues. In this plot study, one conventional and one BMR corn hybrid were planted and subjected to an abundant (60,000 seeds/ha and 225 kg N/ha) and a limited (90,000 seeds/ha and 180 kg N/ha) environment. The ruminal NDFD was determined in vitro in leaf blades, leaf sheaths, and stem internodes. Cell walls from BMR corn had greater NDFD than cell walls from conventional corn on most tissues. Relative to the abundant environment, the limited environment had minimal effects on NDFD. As maturity advanced, NDFD decreased for various but not all tissues. In conclusion, under the conditions of this study, intrinsic characteristics of corn, such as genotype and maturity at harvest, had a greater effect on NDFD than environment or agronomic management.

This study aimed to characterize the effects of increased milking frequency (IMF) at early and mid-lactation on milk yield and its association with changes in cistern and alveolar capacity. Fourteen multiparous Holstein cows were subjected to IMF using the unilateral frequent milking method from 3 to 24 d in milk (DIM). At mid-lactation, cows were randomly assigned to 1 of 2 treatments: control or repeated. From 150 to 170 DIM, IMF treatment was reimposed in the repeated group. During IMF, left udder halves were milked 2× and right udder halves were milked 4× daily. To separate individual milk yields of udder halves, separate buckets were used to collect samples from each udder half. Milk samples and milk yield from right and left udder halves were collected on d 150, 170, 200, 230, 260, and 290 of lactation. Alveolar and cistern capacity were measured 26 h after the last milking at 140 and 172 DIM using an oxytocin inhibitor. Cistern and alveolar capacity were measured by evaluating the milk harvested after oxytocin inhibitor and oxytocin administration, respectively. Udder half difference yields were calculated by subtracting left half yield from right half yield. At 170 DIM, the udder half difference in repeated was 2.27 kg greater than the udder half difference in control. Udder halves milked 4× produced more milk and protein than 2× udder halves in the repeated group at 170, 200, 230, and 260 DIM. Cumulative (150 to 290 DIM) and carry over (200 to 290 DIM) udder half differences in milk yield were similar between the control and repeated treatments. Alveolar volume was similar between udder halves milked 2× or 4× at 140 DIM, while cistern volume was larger for udder halves milked 4× than 2× in early lactation. There was no difference between alveolar or cistern volume proportion in udder halves milked 2× or 4× before mid-lactation IMF. After 20 d IMF for the repeated group, alveolar volume was similar between control and repeated independent of udder half milking frequency. However, repeated held 4.9 kg more cistern milk than control. Control treatment udder halves had a greater alveolar proportion than repeated treatment udder halves. As expected, the cistern proportion was smaller in control and larger in repeated after mid-lactation IMF. IMF at early and mid-lactation enhances milk and protein yield largely during differential milking frequency regimens. The lack of enhancement in milk yield after IMF might be associated with a different response to IMF in the mammary gland at early versus mid-lactation. Based on our results, we conclude that udder halves subjected to early and mid-lactation IMF had increased cistern volume capacity.

Polyunsaturated fatty acids (PUFA) play a role in regulating the body's response to inflammation. Most grains fed to horses are high in linoleic acid (LA), a pro-inflammatory n6 PUFA, compared with α-linolenic acid (ALA), an anti-inflammatory n3 PUFA. Recent interest in hemp (Cannabis sativa) seed oil (HSO) as a PUFA source has arisen due to its unique fatty acid (FA) profile, which includes γ-linolenic acid (GLA), an n6 PUFA with anti-inflammatory properties. Dietary GLA is rapidly converted to dihomo-γ-linolenic acid (DGLA), a precursor to anti-inflammatory eicosanoids. Manipulating dietary FA may lead to alterations in tissue FA profiles in the horse which could promote a reduced inflammatory response. Thus, our objective was to determine if oral supplementation of HSO for 28 d would cause detectable changes in FA composition in synovial fluid (SF) and skeletal muscle (MUS). Six Thoroughbred geldings (11 ± 3.2 yrs, 568 ± 26 kg BW) were used in a crossover experiment with 2 consecutive 63d periods. Horses were offered a control (CON) basal diet of hay and concentrate or the basal diet with the addition of 166 mL HSO delivering 5g GLA. Diets were designed to be isocaloric. Over 7d, HSO was introduced gradually, maintained for another 28d, and then removed and horses resumed basal diets for an additional 28d. Horses were weighed, and body condition score (BCS) assessed weekly. MUS and SF samples werecollected on d0, d35, and d63 of each period. MUS biopsies were taken from the middle gluteus muscle at a depth of 8cm. SF was collected from the left carpus joint. FA were extracted from MUS and SF and analyzed by GC. Individual FA are represented as g of FA per 100g of total fatty acids. Data were analyzed using Proc MIXED in SAS (v.15.1 SAS Institute Inc., Cary, NC). No changes in BW or BCS were observed throughout the study. In SF, GLA was detected after 28d of HSO supplementation (0.32 ± 0.06g/100g) but not at any other time point. On d 28, horses supplemented with HSO also had greater SF DGLA (0.31 ± 0.04g/100g) than CON (0.19 ± 0.04g/100g; P = 0.04). MUS ALA tended (P = 0.07) to be greater in horses fed CON compared with HSO. No other differences were observed for FA in MUS or SF. These results indicate that 28d of HSO supplementation can modify FA profiles in equine skeletal muscle and synovial fluid. This could potentially influence inflammation signaling molecules and subsequently the inflammatory response, however, longer studies with more horses are needed to determine peak incorporation and the effects of varying quantities of HSO in the diet.

LinProTM (LP) is a commercial dietary supplement marketed to increase hoof growth and quality. Ten mature (5-15 years) non-pregnant Quarter Horse mares without existing hoof quality issues were used to test the hypothesis that 32 weeks of daily supplementation with 113 g of LP would increase hoof growth rates as compared to non-supplemented controls. Hooves were trimmed at the start of the study and every 8 weeks thereafter. A mark was applied on the hoof wall surface at 2.5 cm below the junction of the hoof wall and coronary band. At each trimming, the distance between the mark and coronary band was measured and a new mark placed. For front hooves, horses assigned to LP had greater total hoof growth over 32 weeks (2.65 +/- 0.15 vs. 2.18 +/- 0.12 cm; P = .048) and tended to have greater hoof growth per 8 weeks trimming cycle (0.64 +/- 0.03 vs. 0.55 +/- 0.03 cm; P = .085) than horses assigned to non-supplemented controls (CON). Horses assigned to LP had greater plasma biotin concentrations (2158 +/- 69 vs. 636 +/- 62 ng/L; P < .001) and proportions of erucic acid in hoof tissue (1.03 +/- 0.08 vs. 0.76 +/- 0.07 %; P = .049) as compared to CON. Further, the most abundant fatty acids in hoof tissue were stearic, palmitic, oleic, and linoleic acids. LinPro may provide an effective treatment to improve hoof growth rates in horses with otherwise healthy hooves.(c) 2022 Elsevier Inc. All rights reserved.

Somatic cells normally found in milk are generally either immune cells such as lymphocytes, monocytes and granulocytes, or mammary epithelial cells. The number and composition of somatic cells in milk can be influenced by a variety of factors, including infection and temperature-humidity index. The objective of this study was to determine the specific effects of heat stress on the cellular composition of the somatic cell population in milk. We used flow cytometry to ascertain the concentration and viability of mammary epithelial cells, T cells, monocyte/macrophage, and granulocytes in milk from cows maintained under heat stressed conditions compared to thermoneutral conditions. We found a significant 10% increase in the natural log concentration of epithelial cells in the milk of heat stressed cows compared to thermoneutral cows (9.3 vs. 8.4 ln(cells/mL, p = 0.02)). We also found a 12% decrease in the log concentration of live CD45+ cells (p = 0.04), and a 17% decrease in the log concentration of live CD45+ granulocytes (p = 0.04). No changes were found in CD3+CD45+ cells or CD14+CD45+ cells, however, we noted an unusual population of CD14+CD45− cells that showed significant increases of 10% (p = 0.03) and 12% (p = 0.01) in the log concentration of total and dead cells, respectively, under heat stressed conditions. These results suggest that heat stress influences the relative populations and viability of some somatic cells populations in milk. Increased losses of secretory epithelial cells into milk could have implications for milk production, and fewer viable immune cells could negatively impact the immunocompetence of dairy cows under heat stress.

The objective of this study was to determine the effect of drought stress on neutral detergent fiber (NDF) and lignin (LIG) concentrations and on in vitro dry matter digestibility (IVDMD) and in vitro neutral detergent fiber digestibility (IVNDFD) of leaf blades and stem internodes of corn for silage. Eight plots were blocked (i.e., 4 blocks) and randomly subjected to a watered (W) or non-watered (NW) treatment. Within each block, plots were split into 7 sub-plots, to which 1 of 7 corn hybrids were randomly assigned. Before planting, all plots were irrigated with 150 mm of water to ensure a consistent emergence of corn seedlings. After this pre-planting irrigation, NW plots were not irrigated ever again. After planting, W plots were irrigated with 225 and 360 mm of water pre-tasseling and post-tasseling, respectively. Stem internodes and leaf blades from the second phytomer below (LOWER) and the second phytomer above (UPPER) the ear insertion were collected to determine tissue composition and digestibility. Drought stress increased the concentration of NDF in both leaf blades (628 vs. 613 mg NDF/g DM) and stem internodes (625 vs. 572 mg NDF/g DM). Drought stress decreased IVDMD in stem internodes (0.575 vs. 0.525 IVDMD) but had no effect on IVDMD of leaf blades (0.561 IVDMD). Similarly, drought stress decreased IVNDFD in stem internodes (0.422 vs. 0.391 IVNDFD) but had no effect on IVNDFD of leaf blades (0.536 IVNDFD). Drought stress increased the concentration of lignin in the cell wall of leaf blades (161 vs. 141 mg/g CW) but had no effect on stem internodes (266 mg/g CW). Under the conditions of this study, water supply had a minimal effect on lignin concentration in the cell wall and did not increase the in vitro digestibility of fiber in corn for silage. The latter observation is contrary to the general industry belief that water stress increases fiber digestibility in forages.

Increasing milking frequency (MF) increases milk yield (MY) and farm profit, and optimal milking intervals (MI) prevent milk production decline. The objective of this experiment was to compare the MY effect of even and uneven 4 times daily (4×) MI in early lactation under increased MF. Fourteen multiparous and 6 primiparous cows were milked using unilateral frequent milking, with right udder halves milked 4× and left udder halves milked 2 times daily (2×) for 20 d in early lactation starting on d 5 postpartum. Ten (7 multiparous and 3 primiparous) cows were allocated evenly based on parity and assigned to either the even or the uneven MI groups distinguished by intervals of 9:3:9:3 h or 6:6:6:6 h. The left and right udder halves were milked at 0100 and 1300 h. The right udder glands were additionally milked at 0400 and 1600 h for the uneven MI group and at 0700 and 1900 h for the even MI group. Milk from each udder half was weighed and sampled for components on the final day of treatment and at 60, 120, 180, 240, and 300 d in milk. The overall effect of 4× milking on the right udder halves was a 5.96 ± 0.70 kg/d increase in MY on d 21 of unilateral frequent milking compared with the 2× udder halves. This elevated MY continued through 300 d in milk and averaged 1.56 ± 0.70 kg/d. Increased MF in early lactation increased the udder half difference in total yield throughout a 300-d lactation by 508 kg for milk, 25 kg for milk fat, and 15 kg for milk protein. Increased MF in early lactation increased milk component yields, but there were no differences between MI groups. The lack of treatment difference may be beneficial to farmers. The ability to achieve the same increased MY effect with uneven MI may optimize labor efficiency because early-lactation cows could be milked at the beginning and end of milking sessions. Farmers would not have to add additional milking sessions to achieve the enhanced MY response regardless of normal milking session length.

Maternal supplementation of omega-3 fatty acids during gestation has a variety of benefits on the dam and resulting offspring in several species, including reduced latency to stand and suckle. The objective of the study is to test the hypothesis that foals born to mares supplemented with marine-derived algae during late gestation will have greater plasma DHA concentrations than foals born to either control mares or mares supplemented with flaxseed-derived ALA. The current study utilized 13 American Quarter Horse mares and their foals, assigned to one of 3 dietary treatments: a control basal diet (CON, n = 5), the basal diet plus 242.4 g of a supplement providing marine-derived omega-3 fatty acids (DigestaWellⓇ Omega; MAR, n = 5), or the basal diet with the addition of 190 g of a flaxseed supplement (Smartpak Simple Flax; FLAX, n = 3). Supplements were fed twice daily and were designed to deliver 40 g of omega-3 fatty acids. Mares began treatments 30 d before expected foaling dates and continued through d 5 post-parturition (PP). Plasma was collected from mares before supplementation, and on d 0, 5, and 30 PP. Milk samples were collected from mares at d 0 and 5 PP. Plasma samples were collected from foals at birth, and on d 5 and 30 PP. Fatty acid compositions of samples were determined using lipid extraction and gas chromatography. Data were analyzed using MIXED procedure of SAS, using repeated measures for milk and plasma. Simple effect differences were detected via Tukey test, and α = 0.05. The CON diet provided 29 g of α-linolenic acid (ALA), 0 g of eicosapentaenoic acid (EPA), and 4.5 g of docosahexaenoic acid (DHA), the MAR and FLAX diets provided 27 and 34 g of ALA, 0 and 0.03 g of EPA, and 17.4 and 4.5 g of DHA to mares, daily. Fatty acids are represented as g of fatty acid per 100 g of lipid. DHA (1.9 ± 0.2 g/100g) and EPA (0.3 ± 0.1 g/100g) contributed to a greater percent of plasma lipids in foals born to MAR supplemented mares compared with CON foals (DHA = 0.6 ± 0.2; EPA = 0 g/100g; P < 0.05). These fatty acids were not different between FLAX and CON foals (P > 0.1) Samples obtained from foals at birth before nursing had the highest DHA levels (1.90 ± 0.18 g/100g), compared with all other samples, and DHA declined at d 5 (0.6 ± 0.2 g/100g) and 30 (0.1 ± 0.2 g/100g; P < 0.001) PP. No differences were observed in mare plasma or milk. Maternal supplementation of marine-derived omega-3 fatty acids in late gestation increased foal plasma DHA concentrations more so than that of foals from mares fed flax.

Trans-10,cis-12 conjugated linoleic acid (t10,c12 CLA) is well recognized as a key CLA isomer responsible for the reduction in milk fat synthesis that leads to milk fat depression in dairy cows. Sterol regulatory element binding protein-1 (SREBP1) is a key transcription factor in bovine mammary gland coordinating transcription of the genes for fatty acid synthesis. SREBP1 activation requires the removal of insulin-induced gene-1 (Insig1) that serves as a repressor of SREBP1 in the endoplasmic reticulum (ER). We hypothesized that t10,c12 CLA reduced SREBP1 activation by delaying Insig1 degradation. In the present study, we used undifferentiated bovine mammary epithelial cells (MAC-T cells) and treated them with t10,c12 CLA for 6 h. We found that SREBP1 protein expression declined over 56% when cells were treated with 60 µM or greater concentration of t10,c12 CLA. Such inhibitory effects were also observed in the mRNA expression of SREBP1-regulated genes including SREBP1, fatty acid synthetase, stearoyl-CoA desaturase, and Insig1. Compared with no CLA group, 60 µM or higher concentration of t10,c12 CLA increased Insig1 protein expression over 2-fold in cells transfected with FLAG-tagged Insig1. This stimulatory effect was not specific to t10,c12 CLA but also other polyunsaturated fatty acids including cis-9,trans-11 CLA and linoleic acid. Oleic acid had no effect on Insig1 protein expression, whereas palmitic acid decreased Insig1 protein expression. Further investigation revealed that increased abundance of FLAG-Insig1 with t10,c12 CLA was due to the inhibition of the proteasomal degradation of Insig1. The t10,c12 CLA delayed the Insig1 decay when protein synthesis was blocked. Immunoprecipitation also confirmed that the interaction between ubiquitin-like domain-containing protein 8 and Insig1, the key step of removing Insig1 from ER and freeing SREBP1 for proteolytic processing, was inhibited by t10,c12 CLA, but not palmitic acid. These findings suggested that t10,c12 CLA played a role in regulating SREBP1 activation by reducing proteasomal degradation of Insig1. We concluded that stabilized Insig1 retained SREBP1 in the ER from activation, thus reducing lipogenic gene transcription.

Sampling frequent time points of mammary signaling pathways is not possible with tissue biopsies. We have validated a flow cytometry and cell sorting procedure for isolating live bovine mammary epithelial cells from somatic cell populations in milk using butyrophilin 1A1 as a marker for mammary epithelial cells and CD45 as a marker for hematopoietic cells. Hoechst 33342 staining and propidium iodide exclusion were used to select for nucleated live cells. Positive selection of butyrophilin (BTN)-expressing cells was performed by fluorescence-activated cell sorting. Quantitative real-time PCR performed on mRNA isolated from these cells showed a 226-fold increase in κ-casein (CSN3) mRNA expression in BTN single-positive cells compared with unsorted cells, whereas CD45 single-positive cells showed a significant decrease. A negative selection strategy for cells not expressing the hematopoietic cell marker CD45 also resulted in a cell population with a 196-fold increase in CSN3 mRNA expression compared with unsorted cells. We found no enrichment of CSN3 mRNA expression after sorting cells using cytokeratin antibodies. The noninvasive assays described here allow for daily or more frequent sampling time points for measurement of mammary epithelial cells during the course of lactation.