Effects of lipid supplementation on the yield and composition of milk from cows with different beta-lactoglobulin phenotypes.

Responses to lipid supplementation differ between dairy breeds and genetic lines suggesting nutrition by genotype interactions. beta-Lactoglobulin phenotype is associated with changes in yield and composition of milk. The response of cows with different beta-lactoglobulin phenotypes to lipid supplementation has not been examined. Furthermore, we examined whether lipid supplementation alters milk protein composition. By using a randomized block design, we fed Holstein cows for 3 wk either a control diet containing 2.8% crude fat (n = 19) or an experimental diet that was supplemented with 4.2% tallow (n = 20). Before randomization, all cows were fed the supplemental tallow diet for at least 2 wk. Dry matter intake, body weight, milk yield, and milk composition were measured in the last week before and during the experimental period. Feeding supplemental tallow increased dry matter intake and yields of milk and milk components, including casein content, without decreasing milk component content or altering milk protein composition. On the low-fat control diet, cows with the beta-lactoglobulin allele B had a greater milk and milk component yield than cows with the A allele, whereas no differences by beta-lactoglobulin phenotype were observed in cows on the tallow supplement diet. Our results suggest that cows that differ in beta-lactoglobulin phenotype respond differently to a low-fat diet and that feeding cows 4.2% of additional tallow increases milk yield without affecting milk component content and milk protein composition.

Effects of glucagon infusions on protein and amino acid composition of milk from dairy cows.

Changing the composition of milk proteins and AA affects the nutritional and physical properties of dairy products. Intravenous infusions of glucagon decreases milk protein production and concentration by promoting the use of gluconeogenic blood AA for hepatic glucose synthesis. Little is known about how the diversion of AA to gluconeogenesis affects the composition of milk proteins and AA. The objective was to quantify changes in composition of milk protein and AA in response to i.v. glucagon infusions. Three separate experiments were used: 1) 8 Holstein cows were fed ad libitum and infused with glucagon at 10 mg/d for 14 d, 2) 7 Holstein cows were feed restricted and infused with glucagon at 10 mg/d for 14 d, and 3) 4 Brown Swiss cows were infused with glucagon at 5 and 10 mg/d for 2 d each. Milk and milk component yields and milk protein and amino acid composition of samples, collected with blood samples at the first and last day of the glucagon infusion period, were compared with those collected 1 d before and after the glucagon infusion period. Glucagon infusions decreased milk protein production and concentration in each experiment by at least 0.2 +/- 0.05 kg/d and 4 +/- 0.4 g/L, respectively. The decrease was accompanied by changes in milk protein composition, the most consistent being an increase in kappa-casein (1.68 +/- 0.27%). Overall, glucagon infusions resulted in higher proportions of kappa-casein and alpha(S2)-casein (1.34 +/- 0.51%) and smaller proportions of alpha(S1)-casein (-3.83 +/- 1.75%) and alpha-lactalbumin (-0.91 +/- 0.32%). Glucagon had little impact on milk AA composition except an increase in glycine (0.26 +/- 0.11%). The results suggest that milk protein synthesis is regulated by many factors including AA and glucose availability.

Short communication: estimates of genetic variation of milk fatty acids in US Holstein cows.

Interest in changing the milk fatty acid profile is growing. However, little is known about the genetic variability of milk fatty acids in the US Holstein population. Therefore, genetic parameters for milk fatty acids were estimated using a single-trait, mixed, linear animal model on 592 individual milk samples from 233 daughters of 53 sires in a cow herd genetically representative of the US Holstein population. Heritability (h(2)) and repeatability (r) estimates +/- standard errors for yields of individual fatty acids ranged from 0.00 +/- 0.08 (C4:0) to 0.43 +/- 0.13 (C12:0) for heritabilities and from 0.21 +/- 0.05 (C18:1) to 0.43 +/- 0.05 (C12:0) for repeatabilities. Saturated (h(2) = 0.23 +/- 0.12; r = 0.36 +/- 0.05) and de novo synthesized fatty acids (C6:0 to C14:0; h(2) = 0.30 +/- 0.13; r = 0.40 +/- 0.05) had numerically higher estimates than did monounsaturated (h(2) = 0.09 +/- 0.09; r = 0.22 +/- 0.05) and polyunsaturated fatty acids (h(2) = 0.08 +/- 0.09; r = 0.27 +/- 0.05). For relative proportions of individual fatty acids, the greatest heritability and repeatability estimates were obtained for C8:0 (h(2) = 0.18 +/- 0.12; r = 0.36 +/- 0.05), C10:0 (h(2) = 0.22 +/- 0.13; r = 0.46 +/- 0.05), C12:0 (h(2) = 0.18 +/- 0.12; r = 0.46 +/- 0.05), C16:0 (h(2) = 0.09 +/- 0.12; r = 0.48 +/- 0.05), C16:1 (h(2) = 0.49 +/- 0.13; r = 0.49 +/- 0.05), and C18:0 (h(2) = 0.24 +/- 0.11; r = 0.39 +/- 0.05). Our results suggest the existence of genetic variability of milk fatty acids, in particular of medium-and long-chain fatty acids (C8:0 to C18:0), which could be used to improve the nutritional and textural properties of milk fat by selective breeding.

Short communication: Composition of milk protein and milk fatty acids is stable for cows differing in genetic merit for milk production.

Changing the composition of milk protein and of milk fatty acids alters nutritional and physical properties of dairy products and their consumer appeal. Genetic selection for milk yield decreases concentrations of milk protein and of milk fat. Little is known, however, about how the decrease affects composition of milk protein and milk fatty acids. The objective of this study was to quantify changes in composition of milk protein and of milk fatty acids in cows differing in genetic merit for milk production. Three measures of genetic merit for milk production were used for each cow: genetic line, parent average predicted transmitting ability (PTA) for milk, and cow milk PTA. Composition of milk protein and milk fatty acids were compared in 448 milk samples from 178 cows representing 2 divergent lines of Holsteins that were bred for high or average PTA for milk and combined milk protein and fat yield. High-line cows (n = 97) produced more milk that contained less fat and had higher proportions of alphaS1-casein in milk protein than did average-line cows (n = 81). We additionally obtained from 233 cows (178 cows representing the 2 genetic lines and 55 cows with ancestors from both genetic lines) the parent average milk PTA and cow milk PTA and compared composition of milk protein and of milk fatty acids in 592 milk samples. Cows whose parent average milk PTA was above or equal to the median of the 233 cows produced more milk that contained less protein and less fat and that tended to have greater proportions of alphaS1-casein in milk protein than cows whose average milk PTA was below the median. Similarly, cows with above or equal median milk PTA of the 233 cows produced more milk that contained less protein and less fat and had greater proportions of alphaS1-casein in milk protein than did cows with below-median milk PTA. Milk fatty acid composition was not consistently different between groups. Therefore, selection for milk yield decreased concentrations of milk protein and milk fat but had little effect on composition of milk protein and milk fatty acids.

Replacement of bovine mitochondrial DNA by a sequence variant within one generation.

Inheritance of mitochondrial DNA (mtDNA) in Holstein cattle was characterized by pedigree analysis of nucleotide sequence variation. mtDNA was purified from leukocytes of 174 individuals representing 35 independent maternal lineages, and analyzed for nucleotide sequence variation by characterization of restriction fragment length polymorphism and direct sequence determination. These data revealed 11 maternal lineages in which leukocytes from some individuals seemingly were homoplasmic for the reference mtDNA sequence at nucleotide 364, whereas those from other individuals were homoplasmic for a sequence variant at this position. Both alternative alleles were detected in all branches of these 11 lineages, suggesting that mutation at nucleotide 364 and fixation of the variant sequence occurred frequently in independent events. Thirteen instances were detected of mother-daughter pairs in which leukocytes of each of the two animals seemingly were homoplasmic for a different allele at nucleotide 364, demonstrating the bovine mitochondrial genome can be replaced completely by a nucleotide sequence variant within a single generation. The two alternative sequences seemingly arose de novo at similar frequency, ruling out replicative advantage or other selective bias as the explanation for rapid fixation of mutations at nucleotide 364. Another instance of intralineage sequence variation was detected at nucleotide 5602. This variation was detected in only one of the lineages examined, and evidently arose within three generations.

Molecular analysis of cytoplasmic genetic variation in Holstein cows.

Mitochondrial DNA from Holstein maternal lineages implicated to express cytoplasmic genetic effects on lactation traits was subcloned and screened for molecular polymorphisms. Sixteen of 35 lineages sampled differed from the most common mitochondrial DNA form by at least one restriction endonuclease cleavage site in the 4.3 kilobase segment examined. Variation existed in the region that regulates DNA replication and transcription as well as in transfer and ribosomal ribonucleic acid coding regions of the DNA. The index of nucleotide diversity calculated from polymorphism frequencies indicated that the minimum extent of variation between two random lineages was 1.16 x 10(-4) nucleotide differences per base pair in the segment examined. Presence of a HpaII marker near nucleotide 360 was associated with lower (P less than .001) milk fat percentages. Molecular markers indicated that pedigrees may not be sufficient to separate true cytoplasmic lineages for quantitative genetic analyses. These findings provide a molecular genetic basis for further study of cytoplasmic effects on phenotypic variation in Holstein cattle.

Challenges to dairy cattle management: genetic considerations.

Several new technologies will be used to produce continued genetic change in dairy cattle. These technologies are categorized broadly as 1) improved modeling, selection, and evaluation methods; 2) use of new and improved reproductive technologies; 3) new developments in molecular genetics; and 4) new developments in immunogenetics. Improvements in evaluation will continue as computers become faster and have more storage capabilities. Improved mathematical models that more nearly describe the biology of lactation will maximize estimation of genetic differences and reduce residuals. New reproductive technology could allow reduction of generation intervals two- to fivefold compared with present generation intervals and, combined with genetic markers, could markedly accelerate progress. Health problems in dairy cattle are expected to increase as production increases. Thus, selection for decreased incidences of health disorders will be needed, probably by selection of sires with improved general immunocompetence. Research is in the early stages of application of techniques of molecular genetics to animal breeding. Early uses will allow detection and alleviation of genetic defects. Eventually, marker genes that directly affect production and metabolic pathways that also affect production will be subjected to selection. The ability to foresee new and potentially useful techniques will be determined by scientific advancement of areas in which researchers are engaged; thus, accurate prediction far into the future cannot be expected.

DNA methylation of helper virus increases genetic instability of retroviral vector producer cells.

Retroviral vector producer cells (VPC) have been considered genetically stable. A clonal cell population exhibiting a uniform vector integration pattern is used for sustained vector production. Here, we observed that the vector copy number is increased and varied in a population of established LTKOSN.2 VPC. Among five subclones of LTKOSN.2 VPC, the vector copy number ranged from 1 to approximately 29 copies per cell. A vector superinfection experiment and Northern blot analysis demonstrated that suppression of helper virus gene expression decreased Env-receptor interference and allowed increased superinfection. The titer production was tightly associated with helper virus gene expression and varied between 0 and 2.2 x 10(5) CFU/ml in these subclones. In one analyzed subclone, the number of integrated vectors increased from one copy per cell to nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 x 10(5) CFU to an undetectable level. To understand the mechanism involved, helper virus and vectors were examined for DNA methylation status by methylation-sensitive restriction enzyme digestion. We demonstrated that DNA methylation of helper virus 5' long terminal repeat occurred in approximately 2% of the VPC population per day and correlated closely with inactivation of helper virus gene expression. In contrast, retroviral vectors did not exhibit significant methylation and maintained consistent transcription activity. Treatment with 5-azacytidine, a methylation inhibitor, partially reversed the helper virus DNA methylation and restored a portion of vector production. The preference for methylation of helper virus sequences over vector sequences may have important implications for host-virus interaction. Designing a helper virus to overcome cellular DNA methylation may therefore improve vector production. The maintenance of increased viral envelope-receptor interference might also prevent replication-competent retrovirus formation.

Intrinsic labeling of bovine milk with enriched stable isotopes of zinc.

Bovine milk was labeled intrinsically with enriched stable isotopic zinc for human bioavailability studies. Intrajugular administration of zinc isotopes temporarily increased the plasma zinc concentration of Ayrshire cows by as much as 76%, but milk zinc concentration and the distribution of zinc between casein and whey did not change appreciably. Milk zinc isotopic enrichment reached 105 and 613 atom % excess for 67Zn and 70Zn, respectively within 4-12 hr of zinc administration and decreased gradually over several days. This degree of isotopic enrichment is sufficient for testing bioavailability to infants of intrinsic zinc from milk-based formulas.

Health care cost containment measures and mortality in Hennepin County's Medicaid elderly and all elderly.

In Minnesota, several health care cost containment measures occurred about the time Medicare's Prospective Payment System (PPS) was implemented. These included a moratorium on additional nursing home beds, preadmission screening of nursing home applicants, and rapid growth in HMO (health maintenance organization) enrollment by Medicare recipients. Hospital days per elderly Medicaid recipient decreased by 38 percent for those in nursing homes and by 35 percent for those not in nursing homes from 1982 to 1984. By 1986, hospital days per recipient had decreased 53 and 55 percent, respectively, from the 1982 level. Age-adjusted mortality rates for elderly Medicaid nursing home residents for the period 1977 through 1986 showed an increasing trend after 1982. Estimated age-adjusted mortality rates for the entire County population, which had decreased steadily from 1970 to 1982, rose significantly above the projected rate in 1984, 1985, 1986, and 1987. We conclude that, coincident with the institution of the PPS and other health care cost containment measures, use of hospital care has fallen for all elderly Medicaid recipients, age-adjusted mortality rates among those in nursing homes have increased, and the mortality rate trend for the total Hennepin County elderly population has stopped declining.

Texture of butter from cows with different milk fatty acid compositions.

Milk fatty acid composition and textural properties of butter are known to be affected by the cows' diets. We examined the phenotypic variation in milk fatty acid composition among cows fed the same diet to see if the variation was sufficient to produce butter with different textural properties. Ten cows were selected that tested higher (n = 5) or lower (n = 5) in their proportion of milk unsaturated fatty acids. Milk samples were collected a week after testing, and butter was prepared from the individual samples. Milk and butter samples were again analyzed for fatty acid composition. Butter at 5 degrees C was evaluated by a sensory panel for spreadability and by a texture analyzer at both 5 and 23 degrees C for hardness and adhesiveness. Milk and butter samples from cows with a more unsaturated milk fatty acid composition had a lower atherogenic index, and the butter samples were more spreadable, softer, and less adhesive. Thus, phenotypic variation in milk fatty acid composition among cows fed the same diet is sufficient to produce butter with different textural properties.

Regulation of messenger ribonucleic acid expression for gluconeogenic enzymes during glucagon infusions into lactating cows.

The effects of glucagon infusions on expression of mRNA for enzymes that regulate gluconeogenesis were studied in lactating cows. Normal cows and cows with fatty liver that were susceptible to ketosis were assigned to either glucagon-treated or control groups. Glucagon at 0 or 10 mg/d was infused for 14 d beginning at d 21 postpartum. In normal cows, glucagon infusions increased concentrations of both plasma glucagon and glucose, which caused plasma insulin to increase. Consequently, hepatic phosphoenolpyruvate carboxykinase mRNA decreased during wk 1 of glucagon infusions. Glucagon infusions into cows with fatty liver also increased plasma glucagon and glucose, but concentrations of plasma insulin and hepatic phosphoenolpyruvate carboxykinase mRNA did not change. More phosphoenolpyruvate carboxykinase mRNA was present in the livers of cows with fatty liver than in livers of normal cows. In a follow-up experiment with midlactation cows, 3.5-h infusions of glucagon at 14 mg/d increased plasma glucose and insulin and decreased plasma nonesterified fatty acids and hepatic glycogen. Hepatic phosphoenolpyruvate carboxykinase mRNA was decreased 41%, pyruvate carboxylase mRNA was increased 50%, but fructose-1,6-bisphosphatase mRNA did not change. We conclude that the expression of the hepatic phosphoenolpyruvate carboxykinase gene in normal cows is inhibited by insulin to balance elevated carbohydrate status during glucagon infusions; however, inhibited expression of hepatic phosphoenolpyruvate carboxykinase mRNA probably is not involved in the pathogenesis of lactation ketosis.

Associations among individual proteins and fatty acids in bovine milk as determined by correlations and factor analyses.

Associations among quantities and concentrations of individual milk proteins and fatty acids were determined in individual milk samples from 233 Holstein cows. Correlation coefficients among the six major proteins and the eleven major fatty acids in bovine milk were grouped hierarchically. Factor analyses grouped the milk components into seven families: fatty acids 4:0-6:0, 6:0-16:0, 16:0, 18:0, 16:1 plus 18:1 plus 18:2, all milk proteins and beta-lactoglobulin alone. Correlation coefficients and groupings by factor analyses coincided with shared pathways of synthesis or genetic origins of milk proteins and fatty acids because they are the basis of the correlation coefficients. Hence, the results from correlations and factor analyses could be used to develop hypotheses for the synthesis of milk components and other coordinately regulated physiological processes.

Effect of milk protein genotypes on milk protein composition and its genetic parameter estimates.

The effects of kappa-casein (CN) and beta-lactoglobulin (LG) genotypes on milk protein concentration and composition were estimated for the US Holstein-Friesian population using a single-trait, mixed, linear animal model on 592 individual milk samples from 233 cows. Both milk protein genotypes had no statistically significant effect on the total milk protein concentration; however, substitution of the kappa-CN A allele additively increased the proportion of kappa-CN, and substitution of the beta-LG B allele additively increased the proportion of beta-LG in total milk protein. In response, proportions of the other milk proteins, mainly alpha S1-CN, were decreased. For proportions of alpha S1-CN, kappa-CN, and beta-LG in total milk protein, kappa-CN and beta-LG genotypes explain more than 50 and 25% of the heritability and repeatability estimates, respectively. We concluded that kappa-CN and beta-LG genotypes affect the phenotypic and genetic variation of milk protein composition but do not significantly affect milk protein concentration. A possible explanation for our conclusion is that altered gene sequences in the promoter region of kappa-CN B and beta-LG A, linked closely to the respective genotypes, favor the transcription or translation of their own protein at the expense of the synthesis of other milk proteins, in particular of alpha S1-CN.

Sequence heteroplasmy of D-loop and rRNA coding regions in mitochondrial DNA from Holstein cows of independent maternal lineages.

A mitochondrial DNA (mtDNA) fragment containing the D-loop, phenylalanine tRNA, valine tRNA, and 12S and 16 rRNA genes was cloned and sequenced from 36 cows of 18 maternal lineages to identify the polymorphic sites within those regions and to detect the existence of heteroplasmic mtDNA in cows. Seventeen variable sites were observed within the D-loop and rRNA coding regions of bovine mtDNA within a 2.5-kb span. The hypervariable sites in the D-loop and rRNA coding regions were identified at nucleotide positions 169, 216, and 1594. Heteroplasmic mtDNA (variable mtDNA within a tissue) existed extensively in cows and was detected within the above regions in 11 of 36 cows sequenced. The insertion, deletion, and nucleotide transversion polymorphisms were found only in homopolymer regions. Heteroplasmy was observed frequently and seemingly is persistent in cattle. Though heteroplasmy was demonstrated, most lineages and mtDNA sites showed no heteroplasmy.

Recovery of mitochondrial DNA from blood leukocytes using detergent lysis.

Mitochondrial DNA (mtDNA) was isolated from leukocytes contained in whole blood of cattle. Leukocyte membranes except the nuclear envelope were solubilized in a buffer that contained 1% Triton X-100. After sedimentation of cell nuclei, mtDNA was purified from the cell lysate by organic solvent extraction and ethanol precipitation. Approximately 5 micrograms of mtDNA was recovered from 400 ml of whole blood, a quantity sufficient for routine DNA cloning procedures or for detailed restriction mapping studies. mtDNA isolated with this method is a suitable substrate for several DNA-modifying enzymes. Thus, preparation of mtDNA from blood by detergent lysis provides a noninvasive alternative to tissue biopsy for characterization of mitochondrial genotypes in studies of evolutionary genetics and population dynamics.

Effects of decreased availability of glucose for dairy cows.

After a preliminary trial to establish dose responses to phlorizin, five Holstein cows at 6 wk postpartum were used to test the response of cows in negative energy balance to a sudden decrease in availability of glucose caused by phlorizin. Cows were fed equal amounts of feed twice daily to supply 100% of NRC recommendations for protein and 90% of NRC recommendations for NEL and were in negative energy balance throughout the experiment. Phlorizin at 0, 2, and 4 g/d was injected subcutaneously in equal amounts every 6 h for 48 h and caused excretion of 0, 225, and 337 g/d of glucose in urine. Milk production was not decreased, but percentage of milk fat increased linearly (3.34, 3.56, and 3.70%) with increasing phlorizin. Concentrations of glucose (64.2, 62.6, and 59.4 mg/dl) and insulin (518, 432, and 329 pg/ml) in blood plasma decreased linearly, whereas beta-hydroxybutyrate (6.11, 8.88, and 9.98 mg/dl) and NEFA (181, 220, and 271 mu eq/L) increased linearly. The changes were most dramatic during the final 12 h of the 48-h injection interval. Healthy, early lactation cows in negative energy balance seem to have the capacity to make metabolic adjustments to provide adequate substrates for maintenance and milk synthesis and to compensate for short-term increased needs for glucose.

Characteristics of mammary mitochondria in lines of mice genetically divergent for milk production.

The purpose of this study was to determine the relationship of mitochondrial proliferation and ATP production to milk production in two lines of mice that were genetically divergent for milk production. Milk production differed between high production and low production lines by .8 phenotypic standard deviations as determined by cross-fostered litter weight gain from 1 to 12 d postpartum. Mammary weight, mammary total DNA, and RNA:DNA ratio were greater in glands of high line mice. Mammary DNA and protein, expressed per gram mammary tissue, were similar between lines. Mammary mitochondrial mass per gland differed after six generations of divergent selection. Rates of succinate-supported ATP production and ADP:O of isolated mitochondria differed, but the rate of pyruvate-supported ATP production did not differ between lines. Differences between selection lines in mitochondrial mass and in the efficiency of succinate use for support of ATP production were probable consequences of selection for divergent milk production.

Metabolic responses of lactating dairy cows to 14-day intravenous infusions of glucagon.

Twenty cows were assigned at parturition to two groups to study metabolic effects of continuous intravenous infusions of glucagon. Groups were control cows and cows treated with glucagon at 10 mg/d for 14 d starting at d 21 postpartum. Daily blood samples and nine liver biopsies were taken from d 7 to 49 postpartum. Plasma glucagon increased six- to seven-fold during infusions of treated cows. Plasma insulin was increased heterogeneously by glucagon infusions. Plasma glucose increased 11.5 and 9.0 mg/dl during wk 1 and 2 of glucagon infusions. No other plasma metabolites tested (nonesterified fatty acids, beta-hydroxybutyrate, and urea N) were affected by glucagon infusions. Liver glycogen decreased by d 2 of glucagon infusion but was repleted to preinfusion values by d 7 and increased to 169% of the preinfusion baseline values at 3 d after cessation of glucagon. Milk production decreased transiently during glucagon infusions. Both milk production and milk protein percentage decreased during glucagon infusion, which could imply a decreased availability of amino acids for milk protein synthesis. Feed intakes did not increase during glucagon infusions, which was in contrast to the control group. Results indicated that glucagon infusions caused liver glycogenolysis initially and probably enhanced gluconeogenesis but glucagon did not appear to increase lipolysis from adipose tissue in these early lactating dairy cows.