Preweaning nutrient supply alters serum metabolomics profiles related to protein and energy metabolism and hepatic function in Holstein heifer calves

Lifting the preweaning milk restriction in dairy calves has been causally associated with beneficial effects on growth and future lactation performance. However, the biological mechanisms linking early-life nutrient supply and future performance remain insufficiently understood. Thus, the objective of this study was to characterize growth and the metabolic profiles of calves fed a restricted (RES) and an elevated (ELE) milk supply preweaning. A total of 86 female Holstein Friesian calves were blocked in pairs by maternal parity and received identical colostrum supply within block. Treatments randomized within block consisted of a milk replacer (MR; 24% crude protein, 18% crude fat, and 45% lactose) supplied at either 5.41 Mcal of ME in 8 L of MR/d (ELE) or 2.71 Mcal of ME in 4 L of MR/d (RES) from d 2 after birth until they were stepped down by 50% during wk 7 and fully weaned at wk 8. All calves had ad libitum access to pelleted calf starter (17.3% crude protein, 24.4% neutral detergent fiber, 2.0% crude fat, and 18.2% starch), chopped wheat straw, and water. At 2 and 49 d of age, blood samples were taken for metabolomics analysis. The ELE group by design consumed more milk replacer, resulting in a lower starter intake and a greater body weight and average daily gain. The ELE calves consumed 20.7% more ME and 9.7% more crude protein. However, efficiency of growth was not different between groups. Metabolomic profiling using 908 identified metabolites served to characterize treatment-dependent biochemical differences. Principal component analysis revealed clearly distinct metabolic profiles at 49 d of age in response to preweaning milk supply. Changes in energy (fatty acid metabolism and tricarboxylic acid metabolites), protein (free AA, dipeptides, and urea cycle), and liver metabolism (bile acid and heme metabolism) were the main effects associated with the dietary differences. The ELE group consumed proportionately more glucogenic nutrients via milk replacer, whereas the RES group consumed proportionately more ketogenic nutrients from the digestion of the calf starter, comprising a larger portion of total intake. Associated with the higher growth rate of the ELE group, hepatic changes were expressed as differences in bile acid and heme metabolism. Furthermore, energy metabolism differences were noted in fatty acid and AA metabolism and the urea cycle. The metabolic profile differences between the ELE and RES groups reflect the broad differences in nutrient intake and diet composition and might point to which metabolic processes are responsible for greater dairy performance for cows fed a greater milk supply preweaning.     

Effects of preweaning nutrient intake in the developing mammary parenchymal tissue

Historically, mammary gland growth has been considered isometric the first 2 mo of life and then allometric until peripuberty. However, recent work indicated that the mammary gland might be responsive to nutrient intake preweaning. The objectives of this study were to describe the effects of nutrient intake preweaning on mammary gland development and to investigate cell specific proliferation during this phase of development. Twelve dairy heifer calves were fed either a fixed amount of milk replacer (MR; control, n = 6) or an amount of MR adjusted for BW (enhanced, n = 6). Control calves received a constant amount of a 28% crude protein, 15% fat milk MR per day that was equivalent to 2.8 Mcal of metabolizable energy intake per day; enhanced calves received 0.3 Mcal of metabolizable energy intake per kilogram of metabolic body weight (from 4.2 to 8.4 Mcal of metabolizable energy intake per day). All calves had constant access to water and a 22% crude protein commercial calf starter. Calves were killed at 54 ± 2 d. Control calves consumed 32.6 ± 2.4 kg of MR and 6.7 ± 0.5 kg of calf starter per calf, whereas the enhanced calves consumed 69.5 ± 2.4 kg of MR and 1.9 ± 0.5 kg of calf starter per calf over the 54-d period. Further, to evaluate putative stem cell proliferation, BrdU (5-bromo-2′-deoxyuridine; 5 mg/kg) was injected intramuscularly once per day between 12 to 15 d and again once per day between 24 to 27 d of life. Initial and final body weight for the control and enhanced treatments were 39.2, 61.0, 39.7, and 83.2 kg, respectively. At euthanasia, weights of liver, kidneys, pancreas, whole skinned mammary gland, and mammary parenchyma were measured. The growth rate of each organ was calculated using the concept of allometry as the difference in the change in organ weight as a percentage of body weight. The mammary glands of calves fed the enhanced diet were significantly heavier at euthanasia; when mammary parenchymal weight was analyzed, enhanced calves had 5.9 times greater mammary parenchymal mass, indicating the mammary gland was responsive to nutrient intake before weaning. Allometric growth of the mammary gland was initiated preweaning in the calves fed the enhanced treatment. Further characterization of mammary cells that retained BrdU label revealed no significant differences among the tissue slices analyzed between treatments; however, as calves fed the enhanced diet had more mammary parenchymal mass, if the number of label-retaining cells per counted slide were similar between treatments then the enhanced calves had a larger total population of putative mammary stem cells present in the mammary gland.     

The effects of increasing amounts of milk replacer powder added to whole milk on mammary gland measurements using ultrasound in dairy heifers

The aim of this study was to determine the effect of increasing total solids (TS) in the liquid diet during the preweaning phase on mammary gland development in dairy heifers. The increase in TS was obtained with the addition of milk replacer powder to whole milk. Crossbreed Holstein-Gyr heifers (n = 60) were distributed in 4 treatments with different TS concentrations: 13.5% (n = 15), 16.1% (n = 15), 18.2% (n = 15), and 20.4% (n = 15). The liquid diets were provided from 5 to 55 d of age. From 56 to 59 d of age, the total amount of liquid feed was reduced by half. Heifers were weaned at 60 d and monitored until 90 d of age. Ultrasound mammary gland evaluations were performed weekly between 5 to 11 wk of age, using a B-mode ultrasound equipped with microconvex transducer at a frequency of 6 MHz. In those same weeks, the manual palpation of mammary parenchyma (PAR) was performed. Increased TS concentration of liquid diet during the preweaning period increased body weight of heifers, but did not alter PAR growth and the deposition of adipose tissue in the mammary gland evaluated by ultrasonography. The oval-shaped structure of PAR was altered after 2 mo of age. In the evaluated period, PAR growth was isometric with respect to the body growth rate. Palpation scoring of PAR had a strong correlation with the ultrasound evaluation of the PAR.     

Effect of increasing amounts of milk replacer powder added to whole milk on postweaning performance,reproduction, glucose metabolism,and mammary fat pad in dairy heifers

The aim of this study was to evaluate the effects of increasing the total solids (TS) content of liquid feed, by adding increasing amounts of milk replacer powder to whole milk, on age at puberty and postweaning performance, glucose metabolism, and mammary fat pad of dairy heifers. Crossbreed Holstein × Gyr heifers (n = 58) were distributed into 4 different treatments during the preweaning period. Treatments consisted of liquid feeds with TS content of 12.5 (actual TS = 13.5 ± 0.53%; n = 15), 15.0 (actual TS = 16.1 ± 0.03%; n = 15), 17.5 (actual TS = 18.2 ± 0.14%; n = 13), or 20.0% (actual TS = 20.4 ± 0.24%; n = 15), which were provided up to 59 d old. From 60 to 89 d old, the animals received the same starter offered during the preweaning period in addition to corn silage ad libitum starting at 70 d old. From 90 to 104 d old, the animals were adapted to a total mixed ration. At 105 d old, the animals were distributed in 4 paddocks equipped with electronic feed and water bins and were fed the same total mixed ration ad libitum. A period of adaptation to the electronic feed bins occurred from 105 to 119 d old. Feed and water intake and body weight and body frame development were assessed until puberty. Mammary gland evaluations were performed monthly by ultrasonography from 120 d of age until puberty onset. Puberty onset was determined as plasma progesterone concentration greater than 1 ng/mL in 2 consecutive samples collected 7 d apart. The date of puberty onset was recorded as the collection day of the first of these samples. A glucose tolerance test was performed at 280 d of age. The increased TS content of the liquid feed fed during the preweaning period did not affect dry matter intake, performance, age at puberty, glucose metabolism, or mammary gland fat pad at later stages of rearing process. Conversely, hip width and heart girth increased linearly up to 150 and 240 d of age, respectively, as a result of the increased TS concentration of the liquid feed during the preweaning period.     

Differing planes of pre- and postweaning phase nutrition in Holstein heifers: I. Effects on feed intake,growth efficiency,and metabolic and development indicators

The objectives of this study were to determine the effects of pre- and postweaning planes of nutrition on feed and metabolizable energy (ME) intake, growth, concentrations of glucose, insulin, insulin-like growth factor 1 (IGF-1), and β-hydroxybutyrate (BHB) in blood and rumen volatile fatty acids (VFA) from birth to 25 wk of age in Holstein heifers. Heifer calves (n = 36) were randomly assigned to receive either a low (5 L/d of whole milk) or high (10 L/d of whole milk) preweaning plane of nutrition from 1 to 7 wk of age, and either a low (70% concentrate dry total mixed ration) or high (85% concentrate dry total mixed ration) postweaning plane of nutrition from 11 to 25 wk of age. From birth to 25 wk of age, feed intake was recorded daily, and body measures were obtained weekly. Circulating hormone and metabolite concentrations were measured biweekly and total ruminal VFA, fecal starch, and body condition were assessed monthly. Overall, average daily gain and body weight were greater for heifers offered increased planes of nutrition during both the pre- and postweaning phases. Heifers offered the high preweaning plane had greater milk intake (7.7 ± 0.1 vs. 4.5 ± 0.1 L/d) but lower starter intake (0.3 ± 0.04 vs. 0.7 ± 0.04 kg/d) during the preweaning phase than those offered the low plane. High preweaning plane heifers also had greater ME intake from wk 1 to 7, but less ME intake at wk 9 (5.3 ± 0.3 vs. 6.6 ± 0.2 Mcal/kg) than those offered the low plane. Furthermore, overall glucose (118.8 ± 2.9 vs. 110.1 ± 2.9 mg/dL) and IGF-1 (101.6 ± 3.6 vs. 75.9 ± 3.6 ng/mL) concentrations were greater for high versus low preweaning plane heifers, although circulating insulin and BHB did not differ between preweaning plane groups. However, heifers offered the high preweaning plane had reduced total rumen VFA concentrations compared with heifers offered the low plane in the preweaning phase (47.3 ± 2.0 vs. 55.6 ± 2.1 mM). During the postweaning phase, dry matter intake and ME were consistently greater in heifers offered the high postweaning plane. Overall insulin (2.0 ± 0.1 vs. 1.8 ± 0.1 ng/mL), glucose (97.1 ± 0.6 vs. 92.1 ± 0.6 ng/mL), IGF-1 (178.0 ± 4.8 vs. 155.8 ± 4.8 ng/mL), and BHB concentrations (8.8 ± 0.2 vs. 8.1 ± 0.2 ng/mL) were greater in heifers offered the high than the low postweaning plane. In addition, heifers offered the high postweaning plane had increased VFA concentrations in the postweaning phase (73.4 ± 1.3 vs. 63.9 ± 1.3 mM) compared with heifers offered the low postweaning plane. The results indicated that increasing the pre- and postweaning planes of nutrition along with energy levels positively influenced several indicators associated with heifer development before 25 wk of age. Nevertheless, there was limited interaction in growth and development indicators between the 2 phases.     

Technical note: Mammary gland ultrasonography to evaluate mammary parenchymal composition in prepubertal heifers

Bovine mammary gland development studies are often terminal or involve invasive biopsy procedures. Therefore, noninvasive means of assessing mammary development should be considered as alternative methods in live animals. The objective was to test if mammary ultrasonography can be used as a noninvasive way to estimate mammary parenchyma (PAR) composition in prepubertal dairy heifers with different average daily body weight gains. In the 84 d preceding, the ultrasound exam heifers were maintained in 1 of 3 treatment groups. Individual heifers were fed a high gain (1 kg/d; n = 6), low gain (0.5 kg/d, n = 6), or maintenance (n = 6) treatment diet. To achieve desired body weight gains, heifers were fed differing amounts of the same silage-based diet. Mammary glands of 18 crossbred heifers Holstein:Gyr underwent a single mammary ultrasound exam immediately before heifer slaughter, which took place when heifers weighed 142.0 ± 8.0 kg and were 200 d old. The 4 mammary glands of each heifer were evaluated using a real-time B-mode ultrasound machine equipped with a 6.5-MHz micro-convex transducer. Digital images (8-bit) of glands were obtained and PAR was identified within gland. Average pixel values per unit of PAR area were determined for each gland and analyzed at the level of heifer. Pixel results were interpreted on the basis that lower average pixel values reflect PAR with relatively high amounts of protein as opposed to fat. To help validate that the pixel value within PAR is associated with composition of PAR, pixel findings were compared with histological [number of adipocytes in PAR (Nad) and epithelial area in PAR (Ep)] and biochemical [percent crude protein in PAR (%CP), percent ether extract in PAR (%EE), PAR weight (WPAR), and mammary fat pad weight (WFAT)] composition of PAR in these same heifers. Within PAR, %EE and WFAT were positively correlated with pixel values, whereas %CP, Ep, and Nad were negatively correlated. Parenchyma weight did not correlate with pixel values. Regression analyses (fixed effect log-pixel value; random effect treatment) were used to estimate Nad, Ep, %CP, %EE, WPAR, and WFAT. Sensitivity analysis of regression equations revealed that accuracy of tested equations ranged from 0.77 to 0.93 and precision ranged from 0.56 to 0.82. Concordance correlation coefficients of the equations ranged from 0.41 to 0.76. In conclusion, ultrasonography of PAR can accurately measure and predict PAR composition in prepubertal dairy heifers growing at various rates of gain.     

Long-term effects of ad libitum whole milk prior to weaning and prepubertal protein supplementation on skeletal growth rate and first-lactation milk production

Our objectives were to determine the effects of rapid growth rate during the preweaning period and prepubertal protein supplementation on long-term growth pattern and milk production during the first lactation. Forty-six Israeli Holstein heifer calves were fed either milk replacer (MR) or whole milk (WM) from 4 to 60 d age. Calves had free access to WM or MR for 30 min twice daily and free-choice water and starter mix for the entire day. From weaning until 150 d of age, all heifers were fed the same ration. At 150 d of age the heifers were divided into 2 subgroups, with one subgroup supplemented with an additional 2% protein until 320 d of age. Thereafter, all heifers were housed and fed together until calving. Another cluster of 20 heifers was raised on MR and WM treatments and 3 animals from each nursery treatment were slaughtered at 60 d and 10 mo age to determine effects of nursery treatment on organ and adipose tissue mass. Prior to weaning, the MR heifers consumed 0.12 kg/d more DM than the WM heifers, but metabolizable energy intake was not different. Body weight at weaning and average daily gain during the preweaning period were 3.1 kg and 0.074 kg/d higher, respectively, in the WM treatment than in the MR treatment, with no differences in other measurements. Nursery feeding treatment and added protein had no effect on growth rate in the prepubertal period, but the postweaning difference in BW between the WM and MR heifers remained throughout the entire rearing period. The age at first insemination was 23 d earlier and age at pregnancy and first calving was numerically lower for the WM heifers than for the MR heifers. Adipose tissue weights at weaning were doubled in the WM calves. First-lactation milk production and 4% fat-corrected milk were 10.3 and 7.1% higher, respectively, for WM heifers than for MR heifers, whereas prepubertal added protein tended to increase milk yield. In conclusion, preweaning WM at high feeding rates appears to have long-term effects that are beneficial to future milk production. The positive long-term effects of feeding WM on first-lactation milk production were independent of their effects on skeletal growth. Enhanced milk production observed with WM treatment may be related to the milk supply, paracrine or endocrine effects of fat tissues on mammary parenchyma, or a combination of both factors.     

Effect of dietary protein on prepubertal mammary development in rapidly growing dairy heifers

The objective was to determine whether increased dietary protein would enhance mammary development in prepubertal heifers fed for rapid body growth (1.2 kg/d). Fifty-four Holstein heifers (weighing approximately 134 kg) were assigned to one of three treatments. Heifers were fed a total mixed ration with metabolizable energy at 2.85 Mcal/kg and metabolizable protein at low, standard, or high concentrations (37, 41, or 44 g/Mcal of metabolizable energy, respectively) from 3.5 mo of age until slaughter at approximately 46 d after puberty. Heifers fed low, standard, and high protein gained 1130, 1170, and 1180 g/d, respectively. Dietary protein did not affect age or weight of heifers at puberty or slaughter, withers height gain, or carcass composition. Average mammary parenchymal DNA content for heifers on diets of low, standard, and high protein was 595, 619, and 670 mg/100 kg of body weight, respectively, and was not significantly different. However, for heifers that attained puberty early, those fed low protein had 33% less parenchymal DNA than those fed high protein, even though their body growth and carcass composition were not compromised. We conclude that dietary protein does not have a major effect on mammary development of rapidly grown prepubertal heifers, provided the diet contains adequate protein for normal body growth. But we suggest that feeding low-protein diets increases the risk of impaired mammary development when heifers are fed for rapid growth and attain puberty early and that the new National Research Council guidelines for protein relative to energy seem adequate for optimal mammary development.     

Impact of conventional or intensive milk replacer programs on Holstein heifer performance through six months of age and during first lactation

The objectives were to evaluate the impact of conventional or intensive milk replacer (MR) feeding programs on heifer calf performance through 6 mo of age, age at first calving, and first lactation performance. At 3 (±1 d) d of age, 133 Holstein heifer calves from 3 commercial dairy farms were randomly assigned, within calf source, to a conventional [20% crude protein (CP), 20% fat] or intensive MR (28% CP, 18% fat). Milk replacer treatments and percent solids were 1) conventional nonacidified (CNA), 13.9%; 2) conventional acidified (CA), 13.9%; 3) modified intensive high solids (IHS), 16.7%; 4) modified intensive low solids (ILS), 12.5%; and 5) intensive high solids, high feeding (IHSHF), 16.7%. Calves were individually housed and remained on trial for 56 d. At 2 mo of age, heifers were grouped in pens by treatment with 6 heifers per pen (4 pens per treatment). An 18.1% CP grower concentrate mix (dry matter basis) was fed to heifers that received a conventional MR and a 21.2% CP grower concentrate mix was fed to heifers that received the intensive MR preweaning. Heifers were offered 2.45 kg/d (dry matter basis) of their respective grower concentrate mix for 112 d plus free access to hay and water. At approximately 24 wk of age, heifers were transported to a second-stage grower before returning to their respective farms approximately 1 mo before calving. First-lactation performance was determined using Dairy Herd Improvement Association records. The IHSHF treatment resulted in increased calf body weight and hip height during the preweaning and early postweaning (PEP) period and the postweaning heifer grower (PHG) period as compared with the conventional (CNA and CA) or modified intensive MR treatments (IHS and ILS). Calves receiving the IHS treatment were heavier at d 56 of the PEP period compared with the conventional or ILS treatments; however, this growth advantage was not maintained in the PHG period. Feed cost per kilogram of gain during the PEP period was lowest for CNA and CA, intermediate for IHS and ILS, and highest for the IHSHF treatment. There was no effect of MR feeding program on first-lactation performance; however, heifers that received the IHSHF MR preweaning calved 27.5 d earlier than those fed a conventional MR.     

Lactogenic hormones: binding sites, mammary growth, secretory cell differentiation, and milk biosynthesis in ruminants

Roles of the lactogenic hormones prolactin and placental lactogen in mammary development in ruminants were reviewed. In contrast with other ruminants, failure to detect lactogenic activity in the serum of pregnant cows (in excess of that attributed to prolactin) suggests that placental lactogen may have little direct effect on mammary growth or lactogenesis. However, replacement and ablation experiments using ergocryptine provide definitive evidence that increased periparturient secretion of prolactin is necessary for maximal milk production in cattle. Quantitative microscopy indicates a relative failure of mammary cells in cows with inhibited secretion of prolactin to differentiate structurally. Prolactin induces synthesis and secretion of alpha-lactalbumin in prepartum bovine mammary tissue. Temporary disruption of mammary microtubules immediately prepartum in pregnant heifers reduced subsequent milk production, biosynthetic capacity, and cellular differentiation. For maximal milk production, mammary secretory cells apparently must respond to lactogenic hormone stimulation during the immediate periparturient period. Colchicine may desensitize the mammary epithelium to prolactin action. Membrane binding of radiolabeled human growth hormone to ruminant mammary gland provides a measure of lactogenic hormone binding sites. Specific binding to 600 micrograms of mammary membrane protein was 296% greater in lactating, compared with nonlactating, pregnant (65 days of gestation) ewes. Binding capacity (fmol/mg membrane protein) averaged 275 +/- 57 in mammary membranes from nonlactating, pregnant ewes (100 days gestation, n = 2) and 2,325 +/- 521 in mammary membranes from lactating ewes (n = 6, 14 to 21 days postpartum). Greater understanding of hormonal regulation of the ruminant mammary gland likely will result in development of techniques to produce milk more efficiently and perhaps capability to evaluate production potential of young animals.     

Effects of rumen-undegradable protein on intake,performance, and mammary gland development in prepubertal and pubertal dairy heifers

The objective of this study was to evaluate the influence of different amounts of rumen-undegradable protein (RUP) on intake, N balance, performance, mammary gland development, carcass traits, and hormonal status of Holstein heifers at different physiological stages (PS). Sixteen prepubertal (PRE) heifers (initial BW = 106 ± 7.6 kg; age = 4.3 ± 0.46 mo) and 16 pubertal (PUB) heifers (initial BW = 224 ± 7.9 kg; age = 12.6 ± 0.45 mo) were used in an experiment over a period of 84 d. Four diets with increasing RUP contents (38, 44, 51, and 57% of dietary crude protein) and heifers at 2 PS (PRE or PUB) were used in a 4 × 2 factorial arrangement of treatments in a completely randomized design. Throughout the experiment, 2 digestibility trials were performed over 5 consecutive days (starting at d 36 and 78) involving feed and ort sampling and spot collections of feces and urine. At d 0 and 83, body ultrasound images were obtained for real-time carcass trait evaluation. The mammary gland was ultrasonically scanned at d 0 and every 3 wk during the experiment. Blood samples were taken at d 0 and 84 to determine serum concentrations of progesterone, estrogen, insulin-like growth factor I (IGF-I), and insulin. No interaction between PS and the level of RUP was found for any trait. Apparent digestibility of dry matter, organic matter, and neutral detergent fiber corrected for ash and protein was not affected by RUP level but was lower for PRE compared with PUB heifers. Sorting against neutral detergent fiber corrected for ash and protein (tendency only) and for crude protein was greater for PUB than PRE heifers. Pubertal heifers had greater average daily gain (905 vs. 505 g/d) and N retention (25.9 vs. 12.5 g/d) than PRE heifers. In addition, average daily gain and N retention were greatest at 51% RUP of dietary protein. Mammary ultrasonography indicated no effects of RUP amounts on mammary gland composition, whereas PRE heifers had greater pixel values than PUB, indicating higher contents of fat rather than protein in the mammary glands of PRE heifers. Serum progesterone and IGF-I concentration was affected only by PS, and PRE heifers had greater values of progesterone and IGF-I concentrations than PUB heifers. Serum insulin concentration was unaffected by PS but tended to be higher at 51% of RUP. In conclusion, an RUP level of 51% increases body weight, average daily gain, feed efficiency, and N retention in heifers regardless of the PS. In addition, PRE heifers have a lower sorting ability and reduced intake, total-tract digestibility, and N retention. They also have higher amounts of fat in their mammary glands, even at moderate growth rates.     

Technical note: identification of reference genes for gene expression studies in different bovine tissues focusing on different fat depots

Selection of stable reference genes (REF) is important in real-time PCR data normalization. Bovine tissues such as the mammary gland, liver, muscle, and s.c. fat from the tail head have been thoroughly explored for stable REF, whereas fewer reports exist for other fat depots. Therefore, a suitable combination of REF was tested for different tissues of dairy cattle. Holstein dairy heifers (n = 25) were supplemented (100 g/d) with a control fat (n = 15) without conjugated linoleic acids or with rumen-protected conjugated linoleic acids (n = 10) from the day of calving until slaughter at 1, 42, or 105 d postpartum (n = 5, 10, and 10, respectively). Samples from 6 fat depots (omental, mesenterial, retroperitoneal, s.c. tail head, s.c. withers, and s.c. sternum), liver, semitendinosus muscle, and mammary gland were collected. The REF mRNA were quantified and their stability was analyzed using geNorm(plus). The 3 most stable REF in individual fat tissues and muscle were EMD (emerin), POLR2A (RNA polymerase II), and LRP10 (lipoprotein receptor-related protein 10); in mammary gland were MARVELD1 (marvel domain containing 1), EMD, and LRP10; and in liver were HPCAL1 (hippocalcin-like 1), LRP10, and EIF3K (Eukaryotic translation initiation factor 3). The 3 most stable REF in s.c. fat were EMD, LRP10, and EIF3K; in visceral fat were POLR2A, LRP10, and MARVELD1; and for all 6 adipose tissues were LRP10, EIF3K, and MARVELD1. When the mammary gland was added to the 6 adipose depots, at least 5 REF (LRP10, POLR2A, EIF3K, MARVELD1, and HPCAL1) were needed to reach the threshold of 0.15. Addition of liver to the above-mentioned tissues increased the V value. The data improve the comparison of gene expression between different fat depots. In each case, GAPDH had the lowest stability value.     

Effect of feeding prepubertal heifers with a high oil diet on mammary development and milk production

The objective of this study was to evaluate the effect of feeding prepubertal heifers a diet containing a high level of polyunsaturated fatty acids on mammary development and milk production. A total of 116 Holstein heifers were either fed a conventionally formulated concentrate or a high oil (HO) concentrate, using the same formulation but including 20% soybean oil, from birth to 6 mo of age. After 6 mo of age, all heifers were managed identically. Mammary gland development was evaluated on heifers slaughtered at 4 mo (n = 10) and 12 mo (n = 30) of age. Other heifers were bred when they reached 15 mo of age and milk production and feed intake were recorded every day from wk 4 to 18 of lactation. Feeding the high oil concentrate increased the concentration of linoleic acid in blood plasma (176%) and mammary fat pad (78%) at 4 mo of age and mammary fat pad (93%) at 12 mo of age. At 4 mo of age, mammary development was similar in both treatments. At 12 mo of age, total, parenchyma, and stroma weights of the mammary gland were not affected by treatments. However, lipid content was lower and concentration of DNA was higher in the parenchyma of heifers fed the high oil diet. Nevertheless, total parenchymal DNA and dry fat free tissue content did not reach statistical significance despite the fact that they were, respectively, 15 and 21% higher in HO heifers. Milk production and composition was not affected by treatments. In conclusion, feeding prepubertal heifers with a high oil concentrate slightly improved the mammary development but effects were too small to be translated into better lactating performances.     

Optimizing nutrient ratios in milk replacers for calves less than five weeks of age

In trials 1A and 1B, the objective was to determine whether crude protein (CP) concentration could be lowered from 27% CP if Lys and Met were held constant. Forty-five calves per trial were fed milk replacer (MR) powders that contained 23, 25, or 27% CP (dry matter basis) from whey protein. Each MR powder contained 17% fat, 2.44% Lys, 0.75% Met, and 1.56% Thr by adding l-Lys, dl-Met, and l-Thr, and were fed at 0.681 kg/d. In trial 2, the objective was to estimate an optimal CP-to-energy ratio for 2 different amounts of MR fed. Ninety-six calves were fed 1 of 8 MR powders (dry matter basis): 1) 23% CP fed at 0.545 kg/d, 2) 25% CP fed at 0.545 kg/d, 3) 27% CP fed at 0.545 kg/d, 4) 29% CP fed at 0.545 kg/d, 5) 23% CP fed at 0.654 kg/d, 6) 25% CP fed at 0.654 kg/d, 7) 27% CP fed at 0.654 kg/d, and 8) 29% CP fed at 0.654 kg/d. In each MR, l-Lys and dl-Met were added to achieve a Lys:CP ratio of 0.09 and a Met:Lys ratio of 0.31. Holstein calves initially 2 to 3 d old and 43 ± 1 kg of body weight (BW) from 1 farm were fed MR until weaning at 28 d and were monitored for a total of 56 d. Calves were fed an 18% CP starter and water free choice from d 1 and were housed in individual pens bedded with straw in a naturally ventilated nursery with no added heat. Trials 1A and 1B were analyzed individually as completely randomized designs with repeated measures in a mixed model. Trial 2 was analyzed as a completely randomized block design with a factorial arrangement of 2 rates and 4 CP concentrations with repeated measures in a mixed model. In trials 1A and 1B, preweaning average daily gain (ADG) and feed efficiency declined as CP declined. Postweaning performance did not differ among treatments. In trial 2, preweaning ADG was greater and starter intake was lower at the high MR compared with the low MR feeding rate. Pre- and postweaning and overall ADG increased quadratically as CP increased. Preweaning MR rate interacted with CP; thus, at the low MR rate, providing 3.26 Mcal of metabolizable energy (ME)/d (0.0656 Mcal/kg of BW daily), 51.5 g of CP/Mcal of ME was the optimal ratio in the MR (25% CP, 17% fat, 2.26% Lys, and 0.68% Met) to maximize ADG. At the high ME intake, providing 3.71 Mcal/d (0.0743 Mcal/kg of BW daily), 55.0 g of CP/Mcal of ME was the optimal ratio in the MR (27% CP, 17% fat, 2.44% Lys, 0.75% Met) to maximize ADG.     

Effects of photoperiod on mammary development and concentration of hormones in serum of pregnant dairy heifers

Beginning at 128 d of pregnancy, Holstein heifers were exposed to 16 h light, 8 h dark (long days; n = 10) or 8 h light, 16 h dark (short days; n = 10) until 35 d before calving when they were killed. Photoperiod had no effect on weight or proportion of extraparenchymal fat or parenchyma in the mammary gland or on amount of fat, DNA, or RNA in mammary parenchyma. Serum prolactin concentration was 1.7-fold greater under long than under short days. Concentration of melatonin in serum was 2.4-fold greater during dark than light periods. Duration of elevated serum melatonin concentration in the dark period was longer in heifers given short days, but magnitude of this increase was lower than that in heifers exposed to long days. In a second experiment, peak amplitude and area of the periparturient surge of serum concentration of prolactin were 1.8-fold and 1.7-fold greater, respectively, in six Holstein heifers exposed to long days than in six heifers exposed to short days. We conclude that photoperiod had no effect on mammary development during pregnancy, but relative to short days, long days increased serum concentration of prolactin during pregnancy, including the period of the periparturient surge of prolactin.     

Performance strategies affect mammary gland development in prepubertal heifers

In Brazil, the majority of dairy cattle are Holstein × Gyr (H×G). It is unknown whether excessive energy intake negatively affects their mammary development to the same extent as in purebred Holsteins. We hypothesized that mammary development of H×G heifers can be affected by dietary energy supply. We evaluated the effect of different average daily gains (ADG) achieved by feeding different amounts of a standard diet during the growing period on biometric measurements, development of mammary parenchyma (PAR) and mammary fat pad (MFP), and blood hormones. At the outset of this 84-d experiment, H×G heifers (n = 18) weighed 102.2 ± 3.4 kg and were 3 to 4 mo of age. Heifers were randomly assigned to 1 of 3 ADG programs using a completely randomized design. Treatments were high gain (HG; n = 6), where heifers were fed to gain 1 kg/d; low gain (LG; n = 6), where heifers were fed to gain 0.5 kg/d; and maintenance (MA; n = 6), where heifers were fed to gain a minimal amount of weight per day. Heifers were fed varying amounts of a single TMR to support desired BW gains. Over the 84 d, periodic biometric and blood hormone measurements were obtained. On d 84, all heifers were slaughtered and carcass and mammary samples were collected. At the end, HG heifers weighed the most (181 ± 7.5 kg), followed by LG (146 ± 7.5 kg) and MA (107 ± 7.5 kg) heifers. The ADG were near expected values and averaged 0.907, 0.500, and 0.105 ± 0.03 kg/d for HG, LG, and MA, respectively. In addition, body lengths, heart girths, and withers heights were affected by dietary treatment, with MA heifers generally being the smallest and HG heifers generally being the largest. Body condition scores differed by treatment and were highest in HG and lowest in MA heifers; in vivo subcutaneous fat thickness measurement and direct analysis of carcass composition supported this. The HG heifers had the heaviest MFP, followed by LG and then MA heifers. Amount of PAR was highest in LG heifers and was the same for HG and MA heifers. The percentage of udder mass occupied by PAR was lowest in HG heifers, differing from LG and MA heifers. Composition of MFP was not evaluated. Regarding PAR composition, no differences in ash or DM were found. On the other hand, CP concentration of PAR for HG heifers was lower than that for LG heifers, which was lower than that for MA heifers. Regarding the fat content, HG treatment was higher than LG and MA treatment, which did not differ from each other. In PAR, differences in relative abundance of genes related to both stimulation and inhibition of mammary growth were observed to depend on dietary treatment, sampling day, or both. The same can be said for most of the blood hormones that were measured in this experiment. In this experiment, high ADG achieved by feeding different amounts of a standard diet during the growing period negatively affected mammary development.     

Cellular proliferation and apoptosis in Staphylococcus aureus–infected heifer mammary glands experiencing rapid mammary gland growth

Intramammary infections in nonlactating mammary glands are common and can occur during periods of rapid mammary epithelial cell (MEC) accumulation, which may ultimately reduce total MEC numbers. Reduced MEC numbers, resulting from impaired MEC proliferation and increased cellular apoptosis, are expected to reduce future milk yields. The objective of this study was to measure the degree of cellular proliferation and apoptosis in the epithelial and stromal compartment of uninfected and Staphylococcus aureus–infected mammary glands hormonally induced to grow rapidly. Nonpregnant heifers (n = 8) between 11 and 14 mo of age were administered supraphysiological injections of estradiol and progesterone for 14 d. One mammary gland of each heifer was randomly selected and infused with Staph. aureus (CHALL) while another mammary gland was designated as an uninfected control on d 8 of injections. Mammary tissues were collected on the last day of hormonal injections from center and edge parenchymal regions and subject to proliferation assessment via Ki-67 staining and apoptotic assessment via terminal deoxynucleotidyl transferase dUTP nick-end labeling. Differences in cellular proliferation between CHALL and uninfected control quarters were not apparent, but proliferation of MEC was marginally greater in edge parenchyma than in center parenchyma. Coincidently, CHALL quarters experienced a greater percentage of apoptotic MEC and lower percentage of stromal cells undergoing apoptosis than uninfected control quarters. This study also provides the first insight into the mechanisms that allow the mammary fat pad to be replaced by expanding mammary epithelium as edge parenchyma contained a greater percentage of apoptotic stromal cells than center parenchyma. When taken together, these data suggest that Staph. aureus intramammary infection impairs mammary epithelial growth through reductions in MEC number and by preventing its expansion into the mammary fat pad. These factors during periods of rapid mammary growth are expected to impair first lactation milk yield.     

Effects of fat concentration of a high-protein milk replacer on calf performance

The hypothesis was that calves fed high-fat milk replacers (MR) would have reduced starter intake, digestibility, and average daily gain (ADG). Forty-eight Holstein calves (initially 42.4 ± 1.5 kg of body weight, 2 to 3 d of age; 12 calves/treatment) were fed 0.66 kg dry matter (DM) of MR per calf daily that contained 14, 17, 20, or 23% fat. This MR had crude protein (CP) to metabolizable energy (ME) ratios ranging from 51.6 to 56.7 g of CP/Mcal of ME, which were above and below a previously determined optimum. Calves were weaned at 28 d; postweaning measurements were continued to d 56. A 20% CP starter and water were fed ad libitum all 56 d of the trial. Measurements of digestion were made using chromic oxide as a marker in the MR and starter from fecal samples collected on d 19 to 23 from 4 calves/treatment. Selected serum constituents were measured on d 21. Calves were housed individually in pens bedded with straw within a naturally ventilated barn with no added heat. The average barn temperature was 2°C. Data were analyzed as a completely randomized design using polynomial contrasts to separate differences in the means. Preweaning apparent digestibility of DM, organic matter, fat, nonfiber carbohydrates, Ca, and P and serum amylase concentration were linearly reduced as fat increased from 14 to 23%. Preweaning starter intake responded quadratically to fat, being lowest at 14 and 23% fat. A reduction in digestibility and starter intake contributed to less ADG at the higher fat concentrations in the MR. A 27% CP, 17% fat MR with 55 g of CP/Mcal of ME maximized preweaning ADG when fat concentration was varied to obtain various CP to ME ratios in the MR. Additionally, a 27% CP, 20% fat MR with 53 g of CP/Mcal of ME supported overall ADG similar to calves fed the 17% fat MR but preweaning digestion measurements and serum amylase concentrations were less than in calves fed the 17% fat MR.