|Animal Project Approach|
APPROACH AND RESEARCH PROCEDURES
This project makes use of animals from four distinct herds as illustrated in the following table and briefly described below. Two of these herds also supply animals that are used in the project contributing to NP215 that is also conducted at Fort Keogh. All calves are routinely weighed, calving difficulty scored, and neonatal survival recorded within 24-36 hours of birth.
Approximate inventory of breeding age females (N) and relationship of cattle to objectives/sub-objectives of the proposed project on reducing cost of efficient beef production.
CGC Intercross: Females from this herd are used across several objectives/sub-objectives as illustrated in the figure below. Briefly, Objective 2 calls for harvest of 16 of the approximately 140 CGC intercross heifers that are individually fed each year so as to identify those heifers that are most and least efficient in their conversion of feed to body weight. At calving when the heifers are two years of age, 16 additional heifers will be assigned to Sub-objective 5.B. The individually fed heifers also contribute to Sub-objectives 3.A and 4.A. as replacement females for the CGC intercross herd. These heifers will be maintained in the herd until culled for failure to reproduce or advanced age (> 8 years). Bull calves are pen fed as described in Sub-objective 3.A. Bulls needed for breeding are selected at approximately one year of age and the remainder are subsequently sold as feeder cattle to generate income in support of our partnership with the Montana Agricultural Experiment Station.
Line 1 Hereford: This herd contributes primarily to Objective 6.B through the assessment of phenotypes associated with fitness throughout life. The herd is closed, as it has been since 1934, and selection of replacements focuses on growth to one year of age. Yearling bulls selected for breeding undergo a comprehensive semen evaluation and are used in Objective 3.B. Yearling heifers from the Line 1 Hereford herd are used in complementary grazing systems trials conducted in support of the goals of NP 215. Surplus heifers and bulls are marketed either to industry through an annual sale or as feeder cattle to generate income in support of our partnership with the Montana Agricultural Experiment Station.
Angus-Hereford 1: This herd is used primarily to fulfill Sub-objective 5.A. In addition, Objective 1 makes use of ~12 cannulated heifers that are surplus to the needs for replacement females in the herd. Steer calves are sold at weaning to generate income in support of our partnership with the Montana Agricultural Experiment Station. Semen and(or) bulls used for breeding are purchased from industry.
Angus-Hereford 2: This herd is primarily used in assessing the match between seasonal dynamics of forage production and physiological state of animals in support of the goals of NP 215. In this proposal, we examine effects of nutritional management during late fall on various phenotypes and gene expression of cows calving in late winter and late spring (Sub-objective 4.B). Replacement females are generated within the herd. Semen and(or) bulls used for breeding are purchased from industry or obtained from the Line 1 Hereford herd.
Objective 1: Characterize rumen microbial populations, including cellulolytic microbes, and elucidate dynamics of these populations through the use of metagenomic approaches.
Sub-objective 1.A: Identify new species of rumen microbes through whole genome shotgun sequencing of rumen microbial milieu.
This is non-hypothesis driven research
Experimental Design: Rumen contents will be obtained by total evacuation, as needed, from a fistulated cow maintained on an all-forage diet. DNA from rumen microbes may be separated from host animal and dietary plant DNAs by combining filtration and centrifugation techniques and ultimately by subtraction of known sequences. 16S and 18S rDNA libraries will be prepared and sequenced. In addition, whole genome shotgun sequencing and high performance computing, similar to that used in sequencing the human genome, will be applied to the mixed population of organisms obtained from the rumen of a fistulated cow that has been fed an all forage diet. The Venter Institute will do all sequencing and bioinformatics. Genome assembly and subsequent analysis will parallel methodology established for the analysis of many diverse organisms harvested from the Sargasso Sea. While not specifically collaborating with USDA-ARS, Australia and Canada are also expected to contribute financially to this effort.
Expected Outcomes: The capability will be developed to produce microbial DNA of sufficient quality for sequencing from rumen contents. Discovery of many new species of microorganisms involved in digestion of fibrous plant materials is anticipated. Shotgun sequencing of the ribosomal libraries from the rumen microbial milieu will provide species specific 16S and 18S rDNA sequences which will permit quantization of these species in the rumen. Further, discovery of many new genes is also expected. These discoveries lay the foundation of a new mechanistic understanding of symbiosis between rumen microbial population and host animal leading to efficient feed utilization, reducing environmental impact of beef production, and improving sustainability.
Contingencies: If DNA initially harvested is not of sufficient quality for sequencing then more advanced isolation techniques will be developed, e.g., density gradients and ultracentrifugation.
Collaborations: Dr. Michael Brownstein, Venter Institute
Sub-objective 1.B: Compare rumen bacterial species diversity responses to different diets.
Hypothesis 1.B: Indexes of rumen bacterial species diversity are unaffected by differences between grass hay, corn silage, and high concentrate diets.
Experimental Design: Like-age crossbred heifers (n=4 per treatment) of similar genetic background will be ruminally fistulated and randomly assigned to diets differing in level of concentrate (Tabled below). Diets will formulated so as to provide approximately equal amounts of rumen degradable protein. Animals will be individually fed in Calan gates throughout this experiment. Prior to the imposition of treatments on d 0, all animals will be fed the 30% concentrate diet for a minimum of 60 d. Rumen contents will be collected from each animal by total evacuation on d 0, 4, 12, 24, 48, and 96 thoroughly mixed, sub-sampled (remainder returned), and microbial DNA isolated. Rumen VFA and NH3 will be measured concurrently. Blood samples will also be collected concurrently and assayed for urea nitrogen, glucose, NEFA, insulin, cholesterol, and beta-hydroxybutyrate. A combination of PCR using species specific 16S rDNA (generated in Sub-objective 1.A) and a more general approach using universal 16S rDNA primers and denaturing gradient gel electrophoresis (DGGE) will be used in determining the relative abundance of different bacteria. We will generate custom microarray chips with species specific 16S rDNA oligonucleotides. Probes will be generated by using one of the primers used to generate the 16S rDNA libraries on either bacterial total RNA or genomic DNA. Lanes of the DGGE gels are scored for presence or absence of bands at different migration distances (pixels on the image file) and their relative intensities. Across gels, banding patterns will be standardized with a reference pattern included in all gels. These generated banding patterns will be considered as images of the respective bacterial communities (Fromin et al., 2002). Euclidean distances between profiles will be calculated and summarized using AMOVA implemented in ARLEQUIN (Schneider et al., 2000).Linear mixed models for repeated measures will be used in the statistical analysis of blood metabolites.
Diet ingredients and nutrient composition.
Expected Outcomes: This research will result in preliminary quantification of changes in the rumen bacterial milieu associated with dietary changes. Identification of specific species is limited to the approximately 10% of species that colonize the rumen which are currently known. Identifying these shifts may aid in the discovery of ruminal environments that are more efficient and may also reflect on efficiencies of the symbiotic ruminant host animal, thus reducing feed costs and environmental impact of cattle.
Contingencies:1) Laboratory techniques used in this sub-objective to assess microbial diversity are new to us. Two members of our staff (Dr. Waterman and a technician) spent a week with Dr. Morrison at Ohio State learning the techniques. In the event that problems are encountered in the laboratory, we will again seek Dr. Morrison's expertise in resolving those issues. 2) The combination of using specific and universal 16S rDNA primers should detect different levels of bacterial species, however, there may be microbes not present in the original samples sequenced in Sub-objective 1.A due to different diets or geographical location. These microbes may be detected by universal priming and DGGE. If we encounter this, we will isolate the appropriate bands from DGGE and sequence these to generate species specific 16S primers to investigate these species.
Collaborations: Bovine Functional Genomics Laboratory, Beltsville, MD, Dr. Erin Connor (Lead Scientist), project on efficiency of nutrient use in cattle. Samples of rumen pilli will be collected concurrently with sampling of rumen contents for anticipated gene expression studies to compliment other studies by the Bovine Functional Genomics Laboratory.
Objective 2: Determine rumen microbial and host genetic effects associated with differences in measures of efficiency of heifers developed under divergent planes of nutrition or different diets.
Hypothesis 2: Indexes of bacterial species diversity and gene expression in host heifers are unaffected by level of intake and have no association with efficiency of body weight gain.
Experimental Design: Based on our preliminary data, marked differences in numerous measures of phenotypic performance are generated by feeding heifers either ad libitum or at 80% of ad libitum levels of intake. Each year, approximately 140 CGC (50% Red Angus, 25% Charolais, and 25% Tarentaise stabilized composite) heifers will be individually offered either ad libitum access to a corn silage-hay based diet or offered the same diet at 80% of that offered ad libitum and efficiency with which they gain weight will be determined phenotypically and by prediction of breeding value for residual feed intake. After 140 d on feed and at approximately 1-yr of age, heifers that are most and least efficient on each diet (n=4 per group) will be identified and harvested. Those heifers that are not harvested become replacement females for the CGC intercross herd (also, see Sub-objective 3.A, below).
Rumen contents will be collected from each animal by total evacuation immediately after exsanguination, thoroughly mixed, sub-sampled, microbial DNA isolated. Relative levels of rumen microbial species will be determined as described in Sub-objective 1.B.
Samples of pituitary, hypothalamus, muscle, and liver will be collected immediately after exsanguination and will be flash frozen for subsequent extraction of RNA. Patterns of gene expression in specific tissues from each heifer will be evaluated by oligionucleotide microarrays (GeneChip Bovine Genome Arrays, Affymetrix Inc.). The GeneChip Operating Software (GCOS, Affymetrix) will be used to normalize hybridization signals across microarrays, and to provide a qualitative assessment (absent or present) of expression of each gene in each sample. Normalized intensities of each probe determined to be expressed in all samples from at least one experimental grouping will then be analyzed to detect differences due to diet, efficiency and diet by efficiency classification. When statistical differences (P < 0.01) are identified, thresholds to control for magnitude of difference [log 2 transformation of the ratio of signal intensities for two groupings must be equal or greater than 0.6 (i.e., expression is increased by at least 1.5 fold) or be equal to or less than -0.6 (expression decreased by at least 35%)], and absolute change in signal intensity (must be greater than 10 units) will be imposed for final determination of differences in expression (Roberts and McLean, 2006). A sub-sample of genes exhibiting differential patterns of expression will be evaluated in tissues from additional animals by RT-PCR to confirm results from microarray comparisons.Expected Outcomes: Taken together, results from Sub-objective 1.B, our collaborator's (Erin Connor) characterization of gene expression in the rumen epithelium, and from Objective 2 will provide a unique assessment of the interplay between genotype of the host animal and rumen microflora. Normalized intensities of oligionucleotide probes will also be summarized using Euclidean distances and related to those obtained from the rumen microflora. A similar approach was used by Forestier et al., 2002 (cited in Fromin et al., 2002) to assess the relationship of bacterial communities with the physical and chemical environments in which they lived. Identified genes with putative major effects on feed use will be validated against findings from a collaborator's (Harvey Freetly) independent project and ultimately may be exploited through gene-assisted selection to improve production efficiency.
Contingencies: Laboratory techniques used in characterizing the rumen microflora are new to us. Two members of our staff (Dr. Waterman and a technician) spent a week with Dr. Morrison at Ohio State learning the techniques. In the event that problems are encountered in the laboratory, we will again seek Dr. Morrison's expertise in resolving those issues.
Collaborations: Bovine Functional Genomics Laboratory, Beltsville, MD, Erin Connor (Lead Scientist), project on efficiency of nutrient use in cattle.
Objective 3: Determine phenotypic and genetic relationships of early-in-life measures of feed consumption, growth, and body composition, with subsequent reproduction and lifetime productivity.
Sub-objective 3.A: Estimate variances and covariances of longevity, stayability, number of calves produced, and cumulative production of beef cows with early-in-life expression of growth rate, feed consumption/conversion, and body composition.
Hypothesis 3.A: Ability of beef females to annually become pregnant and raise a calf is genetically and phenotypically independent of early-in-life performance.
Experimental Design: Approximately 500 CGC females will be mated annually for 5 years to CGC bulls so as to produce approximately 30 progeny per sire. Non-pregnant, unsound, and 8-year-old cows are culled, with the reason for disposal recorded.
All calves will be weighed at birth, calving difficulty score and neonatal survival recorded, and be reared by their dams until weaning at approximately 6 mo of age. After weaning, approximately 140 heifer calves will be randomly allocated to be individually offered either ad libitum access to a corn silage-hay based diet or offered the same diet at 80% of that offered ad libitum and efficiency with which they gain weight will be determined. Over the 5 years indicated for the proposed research, we anticipate collecting data from ~700 individually fed heifers by approximately 50 sires. These data will be augmented with similar observations on 397 heifers collected previously. In addition to feed consumed during a 140-d evaluation period, body weight will be recorded every 28-d, attainment of puberty will be monitored using periodic blood samples and visual observation, and longissimus muscle area, subcutaneous fat thickness, and intramuscular fat percentage will be measured by ultrasonic imaging at the beginning and end of the evaluation period. Approximately 110 of these heifers will subsequently be returned to the CGC intercross herd, exposed for breeding as yearlings and annually thereafter. After calving as 2-year-olds, 16 additional females will be removed from the CGC intercross herd and used in Sub-objective 5.B. The remaining females will continue in the CGC intercross herd until culled for failure to reproduce, age, or other identifiable criteria. To increase accuracy in evaluation of the traits expressed early in life, half-sib bull calves will be similarly fed in pens of ~25 head each. On the bull calves, body weight will be recorded every 28-d, attainment for puberty at a given age will be assessed by evaluating a semen samples collected at 11 months of age, and longissimus muscle area, subcutaneous fat thickness, and intramuscular fat percentage will be measured at the beginning and end of the evaluation period using ultrasound.
Analyses of the data will be conducted in two stages. First, longevity/stayability of the dams will be related to performance of their progeny and second, early-in-life measures of progeny performance will be related to their subsequent longevity/stayability. Genetic and phenotypic variances and covariances will be estimated using Bayesian methodology appropriate to the estimation of covariances between data with censoring (longevity), and normally and binomially distributed traits (Korsgaard et al., 2003). Posterior distributions of correlations will be evaluated to establish the probability that the correlations are either greater or less than zero.
Expected Outcomes: Heifers that are harvested in Objective 2 and those transferred to Sub-objective 5.B are selected from the heifers fed individually and surplus to needs for maintaining inventory in the CGC intercross herd. In addition, phenotypes recorded in this facet of the proposed research also contribute to QTL identification under Sub-objective 6.A. The research will result in estimates of phenotypic and genetic correlations of longevity/stayability and cumulative productivity with growth, gross and residual feed intake and feed conversion, and body composition measures of young females. An improved ability to use information available in selection decisions to enhance lifetime productivity and thereby reduce costs of producing replacement females will result from this research. Results will also contribute to the incorporation of stayability and(or) longevity into selection indexes used by the beef industry.
Contingencies: Weather related failure to produce adequate feed/forage resources to maintain the CGC intercross cow herd will result in reduced animal numbers and prolong this work. As calves in this environment are typically weaned weighing less than 500 lbs, it may be necessary for us to collect the initial ultrasound images at about 56 d after weaning. This would provide a period of 84 d over which to measure tissue growth rates.
Collaborations: U.S. Meat Animal Research Center, Clay Center, NE, Harvey Freetly (Lead Scientist), project to improve nutrient management and efficiency in cattle; related to assessment of early life indicators of lifetime performance of beef cows. Also, we are actively seeking a collaborator interested in relating levels of metabolic hormones to measurements of growth rate, feed consumption, and body composition.
Sub-objective 3.B: Determine effects of differences in phenotypes measured prior to the time of making selection decisions on subsequent fertility of bulls.
Hypothesis 3.B: Differences in growth rate, body composition, and semen traits of bulls measured before they can be used for breeding have no effect on the number of their progeny.
Experimental Design: Performance of Line 1 Hereford bull calves (n?90/yr) for growth and carcass traits will be measured using standard testing procedures as prescribed by the Beef Improvement Federation. In addition, measures of weight, height, ribeye area, subcutaneous fat depth, and intramuscular fat content are also collected at weaning. The ration fed will be formulated such that the average growth rate of all bulls evaluated is approximately 1.25 kg/d over the 140-d test period and all bulls will be treated alike during the postweaning period. Consistent with the long-established protocol for selection in the Line 1 Hereford population, bulls will be selected based primarily on their growth to 1 yr of age with secondary consideration given to a subjective evaluation of their conformation. At ~13 mo of age, bulls will have their semen extensively evaluated for morphology and fertility associated traits. Fertility will be assessed by pregnancy rate with single-sire mating and by relative numbers of progeny with multiple-sire mating during a 60-day breeding season. Measures of fertility will be regressed on pre-breeding measures of performance and semen quality.
Expected Outcomes: This research will result in improved methods for identifying bulls with high fertility. Results will contribute to methodology to reduce the number of females culled for failing to reproduce and thus reduce production costs associated with generating replacement females.
Objective 4: Determine if the level of nutrition in utero and prior to puberty results in epigenetic effects on traits associated with production efficiency at later stages in life.
Sub-objective 4.A: Determine effects of feed intake prior to puberty and level of supplementation during mid to late gestation on genetic (co)variance and gene expression of the treated animals and their progeny.
Hypothesis 4.A: Within phenotype and across environments, genetic correlations are equal to one and genetic variances are homogeneous.
Experimental Design: The CGC intercross (50% Red Angus, 25% Charolais, and 25% Tarentaise stabilized composite) cowherd will be used. Approximately 500 females (~110 heifers and 390 older cows) will be exposed for breeding each year. During winter and until calving, one of two levels of supplemental feed will be provided to the cows. As is typical in the region and similar to our management of the CGC intercross cow herd for the previous 5 years, supplemental feed will be provided as alfalfa hay beginning about December first, or earlier if weather and(or) grazing conditions warrant. One group of cows (CF) will be provided 0.36 kg/head/day of crude protein as supplement to native forages. The second group of cows (RF) will be provided 0.22 kg/head/day of crude protein from the same feedstuff. Additional hay will be fed when snow cover precludes grazing. The RF group will be fed 80% of the hay given the CF group. The amount of supplemental feed provided may vary between years because of variations in range condition and forage quality. Cows will be maintained as a single herd from parturition until approximately 8 mo postpartum (~2 mo after weaning) when supplemental feeding generally begins at this location. Blood samples will be collected immediately before and after the supplementation period and after calving for later determination of metabolic status by measuring circulating levels of IGF-1 and other metabolic factors (Roberts et al., 1997).
The highly dynamic nature of the range forage resource makes formulating total experimental diets based on nutrient content difficult. Providing varied levels of protein supplement to the two groups of cows should result in variations in forage intake and digestibility that will impact total energy intake of the beef cow in a manner greater than that of the nutrient content of the supplement alone. Model 1 of the computer program developed by NRC (1996) was used to evaluate potential impacts of limited levels of supplemental feed with varied qualities of range forage. Supplementation programs were then designed to provide optimal and less than optimal crude protein and DIP levels based on average forage quality.Five times per year (before breeding, at weaning, at initiation of supplementation, and before and after calving) cows will be weighed and body condition scored. Except at weaning, a blood sample is also collected from each cow for hormone assays. Hip height and udder score are recorded annually, at weaning and calving, respectively. Performance of the progeny of these cows will be measured as described in Sub-objective 3.A above. Briefly, the progeny performance data collected will include periodic weights, measures of fitness, age at puberty, pregnancy rates, harvested feed inputs, date at and reason for death/disposal.
Within each progeny phenotype, the genetic correlations across nutritional environments will be estimated using REML for animal models as measures of genotype by environment interaction. A measure of the broadly defined epigenetic effect is obtained when the genotype is that of the in utero calf and the environment is defined by maternal nutrition during gestation. Genetic variances will also be calculated for each nutritional environment using Bayesian approaches under the prior belief that the direct genetic variances should be similar (Van Tassell and Van Vleck, 1996). Posterior distributions of the variances will be evaluated to establish the probability that direct genetic variances (i.e., variances attributable to progeny genotype) are conditioned on the level of nutrient intake.
In the event that significant effects on either phenotypic performance or genetic variance are detected, microarray and(or) RT-PCR procedures will be used to compare patterns of gene expression in tissues collected from extreme animals (n = 4 per group). Tissue samples collected will minimally include hypothalamus, pituitary, liver, muscle, rumen duodenum, jejunum and ileum, with additional tissues to be collected driven by differences in phenotypic measures observed. Patterns of gene expression in the selected tissues will be evaluated by oligionucleotide microarrays (GeneChip Bovine Genome Arrays, Affymetrix Inc.). The GeneChip Operating Software (GCOS, Affymetrix) will be used to normalize hybridization signals across microarrays, and to provide a qualitative assessment (absent or present) of expression of each gene in each sample. Normalized intensities of each probe determined to be expressed in all samples from at least one experimental grouping will then be analyzed to detect differences due to diet of the dam. When statistical differences (P < 0.01) are identified, thresholds to control for magnitude of difference [log 2 transformation of the ratio of signal intensities for the two groupings must be equal or greater than 0.6 (i.e., expression is increased by at least 1.5 fold) or be equal to or less than -0.6 (expression decreased by at least 35%)], and absolute change in signal intensity (must be greater than 10 units) will be imposed for final determination of differences in expression (Roberts and McLean, 2006). A sub-sample of genes exhibiting differential patterns of expression will be evaluated in tissues from additional animals by RT-PCR to confirm results from microarray comparisons.
Expected Outcomes: This research will provide practical guidance for beef producers regarding consequences of providing different levels of harvested feeds in development of replacement females and maintenance of the cow herd. Since methodology currently employed in national cattle evaluation assumes homogeneity of genetic variances and if genetic variances are found to be heterogeneous, this result points to potential bias in comparison of animals based on their predicted breeding values. If epigenetic effects (i.e. effects of maternal nutritional environment on progeny BV) are detected, these results will also provide motivation for future study. Microarray studies will clarify direct and epigenetic influences of nutrient intake on gene expression.
Contingencies: Failure to have adequate feed resources to maintain animals will result in reduced animal numbers in the CGC population, prolonging this work.
Collaborators: U.S. Meat Animal Research Center, Clay Center, NE, Harvey Freetly (Lead Scientist), project to improve nutrient management and efficiency in cattle; in assessing our shared hypotheses related to effects of differential nutritional paradigms during gestation on subsequently expression indicators of progeny lifetime production efficiency. Also, we are actively seeking a collaborator interested in levels of metabolic hormones in the blood samples collected from the cows and their relationships with the recorded phenotypes.
Sub-objective 4.B: Determine effects of nutrient intake during gestation on phenotypes of treated animals and their progeny.
Hypothesis 4.B: Maternal nutrient intake during gestation has no effect on phenotypes of treated animals and their progeny.
Experimental Design: Two groups of Angus-Hereford crossbred cows calving within late winter (n ? 80 pregnant cows/yr) or late spring (n ? 80 pregnant cows/yr) will be maintained to provide cows in different physiological states throughout the year. Our purpose is not to compare seasons of calving per se, rather cows from within these herds will be subjected to one of two nutritional environments for approximately 60 days during autumn to early winter, thereby resulting in differing levels of in utero nutrition during the last and first trimester of pregnancy for the late winter and spring calving cows, respectively. Because calves from the late spring calving system treated in utero are still suckling dams in the subsequent autumn, treatments can only be applied in alternate years. Two three-year studies will be conducted in tandem over alternating years so that each treatment lasts a full year. In this manner two 3-year studies can be conducted over 6 years. One study will be conducted to evaluate the impact of nutrient supplementation for late spring calving cows grazing native rangeland. Forty primi- and multi-parous cows from the late spring calving system will be assigned to receive a supplement and twenty cows will serve as a non-supplemented control. Supplements will be individually fed daily resulting in the individual cow as the experimental unit. The supplement will be formulated to increase the protein intake of grazing cows, but will also impact energy intake. A second study will utilize cows from the late winter calving system. Forty cows will graze seeded forage pastures to increase nutrient availability and forty cows will graze native rangeland. Response variables include periodic weights and body condition scores of cows and resulting offspring and age at puberty, pregnancy rates, and longevity of female offspring. Data for each experiment will be analyzed using linear mixed model methodology to determine effects of nutritional environment.In the event that supplementation strategy or forage source affect either treated cows or their progeny, microarray and(or) RT-PCR procedures will be used to compare patterns of gene expression in tissues collected from extreme animals (n = 4 per group). Tissue samples collected will minimally include hypothalamus, pituitary, liver, muscle, rumen duodenum, jejunum and ileum, with additional tissues to be collected driven by differences in phenotypic measures observed. Patterns of gene expression in specific tissues from each calf will be evaluated by oligionucleotide microarrays (GeneChip Bovine Genome Arrays, Affymetrix Inc.). The GeneChip Operating Software (GCOS, Affymetrix) will be used to normalize hybridization signals across microarrays, and to provide a qualitative assessment (absent or present) of expression of each gene in each sample. Normalized intensities of each probe determined to be expressed in all samples from at least one experimental grouping will then be analyzed to detect differences due to diet of the dam. When statistical differences (P < 0.01) are identified, thresholds to control for magnitude of difference [log 2 transformation of the ratio of signal intensities for the two groupings must be equal or greater than 0.6 (i.e., expression is increased by at least 1.5 fold) or be equal to or less than -0.6 (expression decreased by at least 35%)], and absolute change in signal intensity (must be greater than 10 units) will be imposed for final determination of differences in expression (Roberts and McLean, 2006). A sub-sample of genes exhibiting differential patterns of expression will be evaluated in tissues from additional animals by RT-PCR to confirm results from microarray comparisons.
Expected Outcomes: This research will provide practical guidance for beef producers in the Northern Great Plains regarding consequences of alternative nutritional management systems. In addition, it will provide clarification of genetic ramifications of the influence of nutrient intake during gestation on gene expression during postnatal growth and development of progeny.
Contingencies: Failure to haveadequate forage resources to maintain animals during the grazing season will result in reduced animal numbers being available for this sub-objective, prolonging this work.
Collaborations: Work identified herein is undertaken in conjunction with work at Fort Keogh by Grings and Waterman under NP 215 on the matching of nutritional status resulting from grazing and physiological profiles resulting from different times of calving.
Objective 5: Develop and validate appropriate phenotypes for measuring fertility in cattle in order to determine interactions between variation in cow feed efficiency and reproductive performance.
Sub-objective 5.A: Determine factors controlling establishment and maintenance of pregnancy in cows induced to ovulate different sized follicles.
Hypothesis 5.A: Ability to establish and maintain pregnancy as determined either separately or jointly by maternal or oocyte effects is independent of ovulatory follicle size.
Experimental Design: In preliminary studies, we identified size of the ovulatory follicle as a key factor affecting fertility of cows and heifers with a potential difference in pregnancy rate associated with large versus small follicles size approaching 30%. However, whether the reduced pregnancy rate associated with ovulation of the small follicle results from compromised oocytes or maternal insufficiency remains unknown. We propose using single ovulation reciprocal embryo transfer to partition these effects. Appropriate follow up experiments are also proposed to understand the biological basis for the observed result.
Experiment 1: Seven-day old embryos will be transferred from and to cows on day 7 following GnRH-induced ovulation of small or large dominant follicles. Embryos will be transferred from donors to recipients based on the following GnRH-induced ovulatory follicle treatment groups: 1) small to small (negative control, n = 80), 2) large to large (positive control, n = 40), 3) small to large (n = 120), or 4) large to small (n = 120). A conservative analysis of power to detect effects of 10% (i.e., the 30% effect observed in previous work was divided equally among the three potential sources of variation) on pregnancy rate (given the design) was evaluated by simulation and found to exceed 0.6 for each effect given a threshold significance level of 0.1. Morphological stage and quality grade will be determined for all embryos before transfer based on the International Embryo Transfer Society scoring system. Embryos that are dead or degenerating (quality score = 4) will not be transferred. Uteri of recipients will be examined 20 d following embryo transfer and once every other week until 62 d post-transfer by transrectal ultrasonography to determine establishment and maintenance of pregnancy. Blood samples will be collected from all cows in this experiment before initiation of treatment to determine estrous cyclicity, at prostaglandin treatment (2 d before AI), GnRH-induced ovulation (AI for donors and ovulation for recipients), and embryo transfer to measure progesterone and estradiol and at each pregnancy diagnosis to measure progesterone and pregnancy specific proteins. In addition, blood collected at AI will be used to measure metabolic energy balance. Ultrasound will be used to record ovulatory follicle size at prostaglandin and GnRH-induced ovulation and at embryo recovery/transfer to correlate ovulatory follicle growth rate, size, and estradiol production (serum concentration) with corpus luteum size and progesterone concentration. The hypothesis will be tested using logistical regression to ascertain effects of follicle size of donor and recipient, and interaction of respective follicle sizes on successful establishment of pregnancy and subsequent embryonic/fetal loss.
In the event that ovulatory follicle size effects on fertility are mediated by maternal effects we propose experiments 2 and 3. Experiment 2: ovarian follicular waves of postpartum beef cows will be synchronized and ovaries examined with transrectal ultrasonography 48 h after PG to determine dominant follicle size. Follicular fluid and granulosa cells will be aspirated from large (n = 40) and small (n = 40) dominant follicles. Short-term, serum-free bovine granulosa cell culture, which accurately reflects estradiol secretion of bovine granulosa cells in vivo, will be utilized to examine the estradiol producing capacity of the granulosa cells. Concentrations of androstenedione, IGF-1, estradiol, and progesterone will be measured by radioimmunoassay. Effects of follicle size on hormone concentrations will be determined using procedures for analysis of linear mixed models. Experiment 3: Follicular waves of postpartum beef cows will be synchronized and ovulation induced by GnRH resulting in the ovulation of small or large follicles. Cows with a small dominant follicle (n = 60) present on their ovaries at GnRH-induced ovulation will be equally divided to receive vehicle or estradiol cypionate to better characterize the effects of estradiol on uterine progesterone receptor expression. Cows with a large dominant follicle (n = 30) present on their ovaries at GnRH-induced ovulation will receive vehicle. Endometrial biopsies will be collected trans-cervically on days 6, 9, and 12 post GnRH injection because this interval represents the greatest progesterone receptor expression differential in cattle (Schams, 1987; Zollers et al., 1993). Uterine biopsies will be collected from the uterine horn ipsilateral and contralateral to the ovarian corpus luteum. Extraction of total cellular RNA and characterization of changes in mRNA expression will be determined by real-time PCR. This experiment is designed primarily to evaluate the effects of estradiol production by large and small ovulatory follicles on endometrial progesterone receptor expression. Collaborations with other laboratories (including scientists involved in the W112 Western Region Research Committee which focus research on Reproductive Performance in Domestic Ruminants) may enable evaluation of other endometrial proteins. In any event, we anticipate that this research will lead to additional experiments involving endometrial expression at other times during pregnancy establishment.
In the event that effects of ovulatory follicle size on fertility are mediated by oocyte effects, we propose conducting the following experiment. Experiment 4: ovaries will be removed from cows with large (n = 40) and small (n = 40) dominant follicles, the cumulus oocyte complex retrieved from the dominant follicle, and the oocyte denuded of cumulus cells. Oocytes will be pooled, by follicle size, in groups of 3 (six or more large and small follicle groups of 3 oocytes each) and total RNA will be extracted from each pool. Changes in mRNA expression of Follistatin (a marker of oocyte competence Patel et al., 2007) will be determined by real-time PCR. Ultrasound images of the ovaries and blood sampling will be used to verify the physiological status of the cows from which ovaries are collected. The hypothesis will be tested by regression of mRNA expression on average follicle size of the pool.
Expected Outcomes: This research will determine whether the oocyte, maternal environment (uterus, corpus luteum, and (or) hormonal signals), or both leads to reduced pregnancy rates and(or) increased late embryonic/fetal mortality as observed in previous research. This research will increase basic understanding of how follicle size impacts successful establishment and maintenance of pregnancy.
Contingencies: Failure to have adequate feed resources to maintain animals in the Hereford-Angus crossbred cow herd will prolong this work. Failure to achieve anticipated rates of recovery of high quality embryos will also prolong this work.
Collaborations: Dr. Michael Smith and graduate students from the Univ. of Missouri, Columbia, are actively engaged in all phases of this experimentation.
Sub-objective 5.B: Establish relationships between previous nutrition, time post-partum, resumption of estrus, and energetic efficiency in young postpartum beef cows.
Hypothesis 5.B: Glucose sequestration into tissues, milk production, and resumption of estrous by 2-yr-old beef females are independent of their prior nutritional management.
Experimental Design: Each year for three years, sixteen two-year-old lactating CGC females (8 each from the restricted and control management regimes) with similar calving dates will be removed from the herd described in Objective 4.A. Beginning approximately one week postpartum and continuing through 90 d postpartum, body energetic efficiency as measured by a glucose tolerance test and baseline (pre-infusion) concentrations of beta-hydroxybutyrate will be determined at 14 d intervals. Efficiency with which glucose is incorporated into tissues will be quantified by glucose half-life and glucose and insulin area under the curve. In addition, cows will be milked every 14 d on alternative weeks to evaluate the relationship of milk production and glucose utilization in early lactation. Blood will be drawn weekly and assayed for progesterone to determine the resumption of estrous by each female. Weekly extrusa samples will also be collected from 2 rumen cannulated animals to describe forage quality at times of the aforementioned measurements. Cows will be managed as contemporaries and only receive harvested feeds when forage availability or weather conditions compromise the well-being of the cows. Effects on glucose sequestration will be determined using linear mixed models procedures. Interrelationships among the variables measured will be modeled and quantified using path analysis.
Expected Outcomes: This research will 1) provide practical insight into potential consequences of restricting feed intake in development of beef heifers and 2) clarify relationships between energy metabolism and the return to estrus in 2-year-old range beef cows.
Contingencies: noneCollaborations: noneObjective 6: Identify and fine map quantitative trait loci (QTL) affecting feed intake, growth and reproduction.
Sub-objective 6.A: Identify QTL affecting growth, feed intake, and reproduction.
Hypothesis 6.A: Genes or gene complexes at unknown loci segregate in an advanced intercross of Red Angus, Charolais and Tarentaise and are responsible for differences in growth, feed intake, and reproduction.
Experimental Design: This sub-objective documents an ongoing experiment in which an advanced intercross of Red Angus (50%), Charolais (25%) and Tarentaise (25%) germplasm is maintained and an extensive database of phenotypes collected for growth, reproductive, and carcass traits, as described in Sub-objectives 3.A and 4.A. Briefly, available phenotypes include: periodic weights, measures of fitness, age at puberty, pregnancy rates, feed intake, date at and reason for death/disposal of calves (Sub-objective 3.A), and periodic weights, body condition scores, hip height, udder score, and date of and reason for disposal of cows (Sub-objective 4.A). Breeding values (BV) will be calculated for these phenotypes (n = 700 to 7000+, depending on the specific phenotype) and the 1-2% of animals with extreme BV genotyped using the Bovine BeadChip gene chip technology. Microsatellite genotypes will also be determined approximately every 20 centiMorgan throughout the genome for a sample of all calves produced (n?350). Interval and haplotype mapping will be used to identify QTL affecting variation in both the phenotypes and breeding values.
Expected Outcomes: We expect QTL affecting growth, feed intake, and reproduction of beef females will be identified. Joint consideration of these results with similar results obtained by our collaborator (Harvey Freetly) will validate many of the newly discovered QTL. This research narrows the search for major genes affecting these economically important traits and may ultimately contribute to the implementation of marker assisted selection by the beef industry.
Collaborations: U.S. Meat Animal Research Center, Clay Center, NE, Harvey C. Freetly (Lead Scientist), project to improve nutrient management and efficiency in cattle, for validation of QTL segregating in populations of Bos taurus cattle.
Sub-objective 6.B: Identify QTL with dominance effects on fitness.
Hypothesis 6.B: Genotype frequencies are consistent with expectations under Hardy Weinberg equilibrium, given population size. Experimental Design: Closed populations with small effective size become inbred over generations and the frequency of individuals that are homozygous increases and fitness decreases. Natural selection consistently puts pressure on increased fitness and thus, with dominant gene action, acts to reduce the frequency of homozygous individuals. Genomes of Line 1 Hereford cattle, which are currently approximately 30% inbred, will be surveyed using the Bovine BeadChip gene chip technology. At each locus, the conformity of observed genotypic frequencies to their expectation, given allele frequency and pedigree inbreeding, will be tested. Given a bi-allelic system with initial allele frequencies of A = 0.5, a = 0.5, current (i.e. Fx =0.3) expected genotypic frequencies under the null hypothesis are AA = 0.325, Aa = 0.35, and aa = 0.325. Simulation experiments were conducted with the above initial conditions and varying degrees of heterozygote advantage and sample sizes to determine power-of-the-test from 10,000 replicates of the prescribed experimental conditions. For a single locus without heterozygote advantage, false positive detection rates were consistent with expectation. If the null hypothesis is rejected with probability 0.01 and selection coefficient s = 0.4, then observations on 200 individuals would have probability > 0.75 of detecting the effect if it exists (Figure below). However, this criterion will also produce false positive results and further conformation is necessary.
USDA, ARS Fort Keogh Livestock and Range Research Laboratory