K.M. Daniels

Effects of Milk Replacer Composition on Growth, Body Composition, and Nutrient Excretion in Preweaned Holstein Heifers

Twenty-four newborn Holstein heifer calves were fed 1 of 4 milk replacers (MR): control (20% CP, 21% fat; MR fed at 441 g/d); high protein/low fat (HPLF; 28% CP, 20% fat; MR fed at 951 g/d); high protein/high fat (HPHF; 27% CP, 28% fat; MR fed at 951 g/d); and HPHF MR fed at a higher rate (HPHF+; 27% CP, 28% fat; MR fed at 1,431 g/d). Dry calf starter (20% CP, 1.43% fat) composed of ground corn (44.4%), 48% CP soybean meal (44.4%), cottonseed hulls (11.2%), and molasses (1.0%) was offered free choice. Heifers were obtained from a commercial dairy, blocked by groups of 8 in the order acquired, and randomly assigned to treatments within group. Upon arrival at the research farm, heifers were fed the control for 2 feedings. Treatments were imposed when heifers were 4 +/- 1 d of age. Heifers were on study for 61 +/- 1 d. Body weight and body size measures were taken weekly. Four-day total collection of feed refusals, feces, and urine was initiated at 57 +/- 1 d of age. Heifers were slaughtered at the end of the collection period to evaluate body composition. Preplanned contrasts were used to compare control to all, HPLF to HPHF, and HPHF to HPHF+. Heifers fed the control diet consumed more starter than those fed other treatment diets, but their total dry matter intake and apparent dry matter digestibility were lowest. Fecal output was highest in heifers fed the control diet, whereas urine output and urine N excretion were lowest. Nitrogen intake and urine N excretion were greater for heifers fed HPHF+ compared with HPHF but were not affected by MR fat content (HPLF vs. HPHF). Retention (g/d) of N and P was greater in heifers fed all nutrient-dense diets compared with those fed the control diet, but was not improved by increasing fat in the milk replacer (HPLF vs. HPHF) or by increasing the amount fed. Addition of fat to the milk replacer (HPLF vs. HPHF) increased empty body weight fat content without improving average daily gain or frame measures. Increasing the volume fed (HPHF vs. HPHF+) increased growth rate and empty body weight, but HPHF+ heifers were neither taller nor longer and their carcasses contained more fat. Clear improvements in growth and nutrient retention were observed with more nutrient-dense diets, but most of the improvements were seen with the increased protein intake relative to the control MR; adding fat to the high protein MR did not further improve lean tissue gain.

Functional and gene network analyses of transcriptional signatures characterizing pre-weaned bovine mammary parenchyma or fat pad uncovered novel inter-tissue signaling networks during development

BackgroundThe neonatal bovine mammary fat pad (MFP) surrounding the mammary parenchyma (PAR) is thought to exert proliferative effects on the PAR through secretion of local modulators of growth induced by systemic hormones. We used bioinformatics to characterize transcriptomics differences between PAR and MFP from ~65 d old Holstein heifers. Data were mined to uncover potential crosstalk through the analyses of signaling molecules preferentially expressed in one tissue relative to the other.ResultsOver 9,000 differentially expressed genes (DEG; False discovery rate ≤ 0.05) were found of which 1,478 had a ≥1.5-fold difference between PAR and MFP. Within the DEG highly-expressed in PAR vs. MFP (n = 736) we noted significant enrichment of functions related to cell cycle, structural organization, signaling, and DNA/RNA metabolism. Only actin cytoskeletal signaling was significant among canonical pathways. DEG more highly-expressed in MFP vs. PAR (n = 742) belong to lipid metabolism, signaling, cell movement, and immune-related functions. Canonical pathways associated with metabolism and signaling, particularly immune- and metabolism-related were significantly-enriched. Network analysis uncovered a central role of MYC, TP53, and CTNNB1 in controlling expression of DEG highly-expressed in PAR vs. MFP. Similar analysis suggested a central role for PPARG, KLF2, EGR2, and EPAS1 in regulating expression of more highly-expressed DEG in MFP vs. PAR. Gene network analyses revealed putative inter-tissue crosstalk between cytokines and growth factors preferentially expressed in one tissue (e.g., ANGPTL1, SPP1, IL1B in PAR vs. MFP; ADIPOQ, IL13, FGF2, LEP in MFP vs. PAR) with DEG preferentially expressed in the other tissue, particularly transcription factors or pathways (e.g., MYC, TP53, and actin cytoskeletal signaling in PAR vs. MFP; PPARG and LXR/RXR Signaling in MFP vs. PAR).ConclusionsFunctional analyses underscored a reciprocal influence in determining the biological features of MFP and PAR during neonatal development. This was exemplified by the potential effect that the signaling molecules (cytokines, growth factors) released preferentially (i.e., more highly-expressed) by PAR or MFP could have on molecular functions or signaling pathways enriched in the MFP or PAR. These bidirectional interactions might be required to coordinate mammary tissue development under normal circumstances or in response to nutrition.

Effects of Milk Replacer Composition on Selected Blood Metabolites and Hormones in Preweaned Holstein Heifers

We investigated the effects of increasing dietary protein and energy on concentrations of selected blood metabolites and hormones in Holstein heifers. Twenty-four heifers were fed 1 of 4 milk replacer (MR) diets for 9 wk (n = 6/diet): control [20% crude protein (CP), 21% fat MR fed at 441 g of dry matter (DM)/d], HPLF (28% CP, 20% fat MR fed at 951 g of DM/d), HPHF (27% CP, 28% fat MR fed at 951 g of DM/d), and HPHF+ (27% CP, 28% fat MR fed at 1,431 g of DM/d). Heifers were fed twice daily; water and starter (20% CP, 1.43% fat) were offered free choice and starter orts recorded daily. Serum and plasma aliquots from blood samples collected twice weekly after a 12-h fast were analyzed for insulin-like growth factor (IGF)-I, IGF-binding proteins (IGFBP), growth hormone (GH), insulin, glucose, nonesterified fatty acids, triglyceride, and plasma urea nitrogen concentrations. Only plasma glucose, IGFBP-2, and IGFBP-3 were affected by diet. Dietary treatment differences were only noted when the control was compared with the average of the other 3 diets. The addition of fat to the MR (HPLF vs. HPHF) and increased volume of MR (HPHF vs. HPHF+) had no effect on plasma glucose concentration or relative abundance of IGFBP-2 or IGFBP-3. Heifers fed the control diet had less glucose, greater IGFBP-2, and less IGFBP-3 than the average of the other 3 diets. There was a diet by week interaction for IGF-I. Serum IGF-I concentration in control heifers varied in a quadratic manner with a nadir (20 +/- 4 ng/mL) at wk 4, whereas IGF-I increased linearly in heifers on other diets. Both insulin and triglyceride changed over time in a complex pattern (significant linear and quadratic contrast effects). The greatest concentrations were measured at wk 0.5 with nadirs at wk 6 for both insulin and triglyceride. Serum GH concentration decreased in a linear manner from wk 0.5 to wk 9 in all heifers. Relative abundance of IGFBP-2 was quadratic over time with the greatest amount of IGFBP-2 observed at wk 5. With the exception of glucose, IGF-I, IGFBP-2, and IGFBP-3, the blood variables measured were not influenced by treatment. The IGF-I -GH-IGFBP axis requires further study in heifers to deduce effects of nutrition on hypothalamic regulation of metabolism. We expected to see more treatment differences in concentrations of metabolites involved with protein and fat metabolism. It is likely that the diets used in this study were not diverse enough in composition to elicit such changes or that the efficiency of use of absorbed protein and fat was not different in these animals.

Effects of Feeding Prepubertal Heifers a High-Energy Diet for Three, Six, or Twelve Weeks on Mammary Growth and Composition

The experimental objective was to determine the effects of feeding prepubertal dairy heifers a high-energy diet for 3, 6, or 12 wk on mammary growth and composition. Holstein heifers (age = 11 wk; body weight = 107 +/- 1 kg) were assigned to 1 of 4 treatments (n = 16/ treatment). The treatment period lasted 12 wk and treatments were H0 (low-energy diet fed for 12 wk, with no weeks on the high-energy diet); H3 (low-energy diet fed for 9 wk, followed by the high-energy diet for 3 wk); H6 (low-energy diet fed for 6 wk, followed by the high-energy diet for 6 wk); and H12 (high-energy diet for all 12 wk). The low- and high-energy diets were formulated to achieve 0.6 and 1.2 kg of average daily gain, respectively. Heifers were slaughtered at 23 wk of age and mammary tissue was collected. A longer duration of feeding the high-energy diet increased total mass of the mammary gland, extraparenchymal fat, and intraparenchymal fat, but did not alter the mass of fat-free parenchymal tissue. When adjusted for carcass weight to reflect differences in physical maturity, the mass of fat-free parenchymal tissue decreased in a linear fashion with a longer duration on the high-energy diet. Total masses of mammary parenchymal DNA and RNA were not different. However, after adjustment for carcass weight, the masses of DNA and RNA decreased as heifers were fed the high-energy diet for a longer duration. The percentages of epithelium, stroma, and lumen, the number of epithelial structures, and the developmental scores of mammary parenchymal tissue were not different among treatments. However, the percentage of proliferating epithelial cells in the terminal ductal units, as indicated by Ki-67 labeling, decreased as heifers were fed the high-energy diet for a longer duration. We concluded that feeding prepubertal heifers a high-energy diet for a longer duration resulted in a linear decrease in both the percentage of mammary epithelial cells that were proliferating and in the mass of fat-free mammary parenchyma per unit of carcass. High-energy feeding hastens puberty and, in this study, decreased mammary epithelial cell proliferation in areas of active ductal expansion. These data are consistent with the idea that feeding heifers a high-energy diet will reduce mammary parenchymal mass at puberty.

Level of nutrient intake affects mammary gland gene expression profiles in preweaned Holstein heifers1

Bovine mammary parenchyma (PAR) and fat pad (MFP) development are responsive to preweaning level of nutrient intake. We studied transcriptome alterations in PAR and MFP from Holstein heifer calves (n=6/treatment) fed different nutrient intakes from birth to ca. 65 d age. Conventional nutrient intake received 441 g of dry matter (DM)/d of a control milk replacer (MR) [CON; 20% crude protein (CP), 20% fat, DM basis]. Calves in the accelerated nutrition groups received 951 g/d of high-protein/low-fat MR (HPLF; 28% CP, 20% fat, DM basis), 951 g/d of high-protein/high-fat MR (HPHF; 28% CP, 28% fat, DM basis), or 1,431 g/d of HPHF (HPHF+) MR. Out of 13,000 genes evaluated, over 1,500 differentially expressed genes (DEG) were affected (false discovery rate <0.10) by level of nutrient intake in PAR or MFP. Feeding HPLF versus CON resulted in the most dramatic changes in gene expression, with 278 and 588 DEG having ≥1.5-fold change in PAR and MFP. In PAR, the most-altered molecular functions were associated with metabolism of the cell (molecular transport and lipid metabolism) with most of the genes downregulated in HPLF versus CON. In MFP, DEG also were primarily associated with metabolism but changes also occurred in genes linked to cell morphology, cell-to-cell signaling, and immune response. Compared with CON, feeding HPHF or HPHF+ did not result in substantial additional effects on DEG beyond those observed with HPLF. The pentose phosphate, mitochondrial dysfunction, and ubiquinone biosynthesis pathways were among the most enriched due to HPLF versus CON in PAR and were inhibited, whereas glycosphingolipid biosynthesis, arachidonic acid metabolism, and eicosanoid synthesis pathways were among the most enriched due to HPLF versus CON in MFP and were inhibited. These responses suggest that, in PAR, doubling nutrient intake from standard feeding rates inhibited energy metabolism and activity of oxidative pathways that partly serve to protect cells against oxidative stress. The MFP in those heifers appeared to decrease production of lipid-derived metabolites that may play roles in signaling pathways within the adipocyte. Overall, results indicated that prepubertal/preweaned mammary transcriptome is responsive to long-term enhanced nutrient supply to achieve greater growth rates before weaning. The biological significance of these results to future milk production remains to be elucidated.

Effects of nutrient intake level on mammary parenchyma growth and gene expression in crossbred (Holstein × Gyr) prepubertal heifers

This study investigated the effects of increased nutrient intake levels on prepubertal mammary parenchyma development in crossbreed (Holstein × Gyr) dairy heifers. Eighteen heifers age 3 to 4 mo were fed 1 of 3 nutrient intake levels (n=6 per treatment) designed to sustain an average daily gain of 0.0kg/d (maintenance, MA), 0.5kg/d (low gain, LG), or 1.0kg/d (high gain, HG). Serum blood samples collected on d 42 and 84 after a 12-h fast were analyzed for triglycerides, leptin, insulin, and insulin-like growth factor 1 (IGF-1). Liver and mammary parenchyma were biopsied on d 42 and harvested on d 84 for gene expression analysis. Parenchyma samples were also used for biochemical and histological analysis. Mammary parenchyma weight was lower in HG than in MA or LG heifers, but mammary extraparenchymal fat was greater in HG heifers than in other groups. Heifers fed the HG diet had a greater fraction of ether extract in their parenchyma than the others and a smaller fraction of crude protein in their parenchyma than MA heifers. Moreover, the HG and LG heifers had greater body fat mass than MA heifers. Nutrient intake level had no effect on the number of intraparenchymal adipocytes. Heifers fed the HG diet had greater serum IGF-1 than the others, and serum insulin was lower in the MA than the HG or LG heifers. Liver GHR, IGF1, and IGFBP3 mRNA expression was higher, but IGFBP2 mRNA was lower in HG heifers than in others. The parenchyma mRNA expression of lipogenic markers, such as CD36, ACCA, FASN, and ADIPOR1, was upregulated by nutrient intake level. Significant nutrient intake × time interactions for lipogenic genes during the experimental period indicated variable gene expression depending on the time point of prepubertal mammary gland development. Overall, our data suggest that enhancing nutrient intake increased body fat accumulation and lipogenesis in the mammary gland to the detriment of parenchyma growth. Moreover, increased lipogenesis in the parenchyma of HG heifers may indicate that fat accumulation occurred because of adipocyte hypertrophy and not differences in adipogenesis. The implications of these results for milk yield needs to be elucidated.

Jersey calf performance in response to high-protein, high-fat liquid feeds with varied fatty acid profiles: Intake and performance1

The objective of this study was to determine whether altering the fatty acid (FA) profile of milk replacer (MR) with coconut oil, which contains a high concentration of medium-chain FA, to more closely match the FA profile typically found in whole milk from Jersey cows, would improve Jersey calf performance. Male (n=18) and female (n=32) Jersey calves were assigned at birth to 1 of 4 liquid diets: (1) pasteurized Jersey saleable whole milk [pSWM; 27.9% crude protein (CP) and 33.5% fat]; (2) 29.3% CP and 29.1% fat MR, containing 100% of fat as edible lard (100:00); (3) 28.2% CP and 28.0% fat MR, containing 80% of fat as lard and 20% as coconut oil (80:20); and (4) 28.2% CP and 28.3% fat MR, containing 60% of the fat as lard and 40% as coconut oil (60:40). Calves were fed their respective liquid diet twice daily during wk 1 through 7 and once daily until weaning (approximately wk 8). Calves had ad libitum access to grain and water, and calves were monitored 1 wk postweaning. Average daily gain and body weight did not differ by treatment. Calves fed pSWM tended to have greater hip height (HH) than calves fed 80:20 (80.5 vs. 79.7 cm). Coconut oil tended to have a quadratic effect on HH, with calves fed 100:00, 80:20, and 60:40 at 79.2, 79.7, and 78.5 cm, respectively. No difference was observed in withers height between pSWM and 80:20. Coconut oil had a quadratic effect on withers height, with calves fed 100:00, 80:20, and 60:40 at 76.6, 77.5, and 76.5 cm, respectively. Change in HH from birth to 9 wk tended to be greater for calves fed pSWM than calves fed 80:20 (0.218 vs. 0.194 cm/d). Calves fed pSWM had higher milk dry matter intake (DMI) than calves fed 80:20 (0.580 vs. 0.518 kg/d). No effect of coconut oil was observed on milk DMI. Grain DMI and total DMI did not differ among treatments. Calves fed pSWM had an increase in days with a fecal score >2 compared with calves fed 80:20 (4.24 vs. 2.00 d). Coconut oil had a quadratic effect on fecal score, with calves fed 100:00, 80:20, and 60:40 scoring 4.00, 2.00, and 3.63 d, respectively. Respiratory score did not differ among treatments. In conclusion, DMI and average daily gain were similar among treatments. However, differences among treatments in skeletal growth and fecal scores are indicative of some possible benefits of medium-chain FA on calf health and performance.

Technical note: p40 antibody as a replacement for p63 antibody in bovine mammary immunohistochemistry

Tumor protein 63 (p63) is a nuclear antigen found in basal epithelial cells. To date, 10 isoforms of p63 have been identified, falling into 2 major groups identified by presence or absence of an N-terminal transactivation domain (TAp63 and ΔNp63, respectively). Literature suggests that ΔNp63 is the predominant form expressed in basal epithelial cells and myoepithelial cells (MYEC). The mouse anti-p63 antibody, clone 4B1E12, has been used as a specific nuclear marker for bovine MYEC. Unfortunately, this antibody is no longer commercially available. A new mouse monoclonal antibody, clone BC28, specific to ΔNp63 (designated p40) has been developed. We hypothesized that the p40 antibody would be an appropriate substitution as a MYEC and epithelial basal cell marker. An array of archived formalin-fixed, paraffin-embedded bovine tissues were subjected to immunohistochemical staining for either p40 or p63, with a subset being dual stained for direct comparison. Positive staining for p40 and p63 was observed in serial sections of mammary, skin, rumen, salivary gland, ureter, and bladder. As predicted, negative staining for p40 and p63 was observed in testis and intestine. Dual staining for p40 and p63 in calf mammary (n = 4), lactating mammary (n = 4), rumen (n = 4), and skin (n = 4) showed nearly 100% agreement. Thus, we established that the mouse monoclonal antibody, clone BC28, is a suitable replacement for anti-p63, clone 4B1E12, as a marker of MYEC and basal epithelial cells in bovine tissues.

Technical note: Infusion, sampling, and vacuum-assisted collection devices for use in ruminally cannulated calves

Calves can be ruminally cannulated at young ages, but equipment size limitations preclude use of an infusion and sampling device in these small animals. Likewise, a procedure to easily evacuate rumen contents in young calves has not been described. Overcoming these technical complications related to assessment of ruminal passage kinetics, nutrient digestion, and volatile fatty acid absorption would aid in future studies advancing our knowledge of dairy calf nutrition. The first objective was to design and fabricate 2 devices (one device for infusion and sampling, and another for vacuum-assisted collection) suitable for use in young ruminally cannulated dairy calves. The second objective was to test the utility of these tools when performing procedures commonly used in ruminant nutrition research. A single weaned 62-d-old ruminally cannulated calf was used to evaluate the ability to infuse a solution of LiCoEDTA and sample rumen contents through the cannula cap over a period of 2 h to assess the rumen liquid passage rate (procedure 1). The device was capable of infusing the LiCoEDTA and sampling the rumen fluid, as evidenced by the presence of elevated Co concentrations in the sampled rumen fluid. Using the fluid samples obtained, liquid passage rate within the calf was estimated to be 40.2% of ruminal fluid/h. The second procedure tested the vacuum-assisted collection device and consisted of evacuating and weighing the rumen contents, which is considered a key preparatory step in washed reticulorumen technique experiments that aim to measure nutrient absorption. In agreement with existing literature, evacuated rumen contents represented approximately 4% of the calfs body weight. In conclusion, custom-built devices for infusion, sampling, and vacuum-assisted collection were efficacious when tested in a 62-d-old ruminally cannulated calf fed a diet of 100% texturized starter (18% crude protein, as-fed). Fellow scientists may employ and further modify these techniques to suit their needs when assessing passage kinetics, nutrient digestion, and volatile fatty acid absorption in calves.

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.