Effects of leptin Arg25Cys on peripheral mononuclear cell counts and antibody response to vaccination in beef cattle.

A single nucleotide polymorphism (SNP) in the leptin gene that results in Arg25Cys has been associated with beef carcass quality and milk composition in dairy cattle. However, leptin (LEP) also plays a role in immune performance and hence it was important to determine whether selection based on this SNP would negatively affect immune cell numbers or antibody production. LEP c.73C>T was assessed for effects on immune cell counts and antibody titres in 27 beef cattle herds (n = 556). A commercial rabies vaccine had been administered to these animals. Prior to being vaccinated, counts of several important mononuclear cells (total and activated B lymphocytes, total and activated T helper and T cytotoxic, WC1 T lymphocytes and monocytes) as well as baseline serum antibody titres were determined for each animal. On day 21, antibody titres were measured and a booster vaccine was administered. Finally on day 42, antibody titres and mononuclear cell types were again counted. Counts of six different cell types were significantly associated with the LEP genotype; however, no consistent patterns were observed between LEP genotype (TT, CT or CC) and peripheral blood mononuclear cell populations. Significant differences in the production of rabies antibodies in response to vaccination were observed relative to LEP genotype. Our results suggest that selection for either the C or T allele would not detrimentally impact on the measured indicators of immune function in beef cattle.

Fishy-egg tainting is recessively inherited when brown-shelled layers are fed canola meal.

Feeding canola meal to brown-shelled laying hens can result in the production of eggs with a fishy odor. This fishy taint is caused by the accumulation of trimethylamine (TMA) in the yolk. Trimethylamine is produced by the bacterial fermentation of choline in the lower gut. Fishy-egg tainting is caused by a SNP in flavin-containing monooxygenase 3 (FMO3 c.984A > T), rendering the hen unable to metabolize TMA into the nonodorous TMA N-oxide. The purpose of this study was to characterize the inheritance pattern of fishy-egg tainting when hens are fed canola meal at levels reflecting maximum use based on conventional formulation of laying hen diets. Additionally, we wished to examine the effect of choline source (choline chloride vs. canola meal) on egg tainting. In the first of 2 experiments, 6 hens per genotype (AA, AT, and TT) were allocated to each of 5 dietary treatments (0, 6, 12, 18, or 24% canola meal) for 4 wk. Three yolks per hen collected in the last week of the trial were analyzed for TMA concentration. There was a significant linear regression (P < 0.05) between yolk TMA concentration and dietary canola meal level for hens of the TT but not the AA or AT genotypes. In the second experiment, 6 hens of the TT (homozygous tainting) genotype were each assigned to 1 of 9 dietary treatments: the 5 diets used in the first experiment plus 4 diets that used choline chloride to match the total choline concentration of the 6, 12, 18, and 24% canola meal diets, respectively. Three yolks per hen were analyzed for TMA concentration. A significant response in yolk TMA concentration was seen with the canola meal diets but not the choline chloride diets. We conclude that fishy-egg tainting is recessively expressed when hens are fed canola meal at levels from 12 up to 24% inclusion. We also conclude that choline chloride, at levels typical of commercial egg production, will not lead to egg tainting.

Feedlot performance and immune function analysis of implanted and non-implanted steers selected for alcohol dehydrogenase 1 C (ADH1C) genotype and fed a low vitamin A diet.

Previous studies have shown that the interaction between limiting vitamin A (VA) and an alcohol dehydrogenase 1 C (ADH1C) variant in beef cattle results in increased intramuscular fat in the longissimus thoracis muscle in one genotype when fed low dietary VA. Although quality grade is important for increased profitability and improving taste characteristics of beef products, limiting VA too drastically can impair animal welfare. The objectives of this study were to determine if this marker-assisted management strategy would be effective, and whether any impairment in immune function would occur in a feedlot setting. Mixed breed beef steers (n=2000) were sorted into 40 feedlot pens so that all combinations of ADH1C genotype (TT or CT), VA level (50% or 100% of recommended) and hormonal implant status (implanted (IMP) or non-implanted (NI)) were equally represented within the population. The VA×ADH1C interaction was not observed. An implant status × ADH1C interaction was observed with average daily gain (ADG; P=0.03). Steers that were IMP and CT had higher ADG than IMP TT (CT=1.69 and TT=1.62 kg/day), whereas both genotypes in the NI steers were lower (CT=1.29 and TT=1.32 kg/day). Implant status was shown to affect dry matter intake (DMI; IMP=8.55 and NI=7.87 kg; P<0.01), total days-on-feed (IMP=164.4 and NI 210.5 days; P<0.01), USDA yield grade (YIELD; IMP=2.40 and NI=2.77; P<0.01), marbling score (MARB; IMP=392 and NI=455; P<0.01), longissimus thoracis area (LTA; IMP=85.0 and NI=80.7 cm2; P=0.01) and backfat thickness (FAT; IMP=8.0 and NI 10.0 mm; P<0.01). Overall, IMP animals finished on fewer total days-on-feed with higher ADG, DMI, larger LTA, and lower YIELD, MARB and FAT. To investigate immune function parameters, crossbred steers (n=18) were selected from a prior feeding trial so that all combinations of ADH1C (TT, CT and CC) and VA (25% or 75%) were equally represented. Blood cell count analysis and peripheral blood mononuclear cell proliferation and stimulation assays were conducted. None of these immune parameters were affected by VA level. Treatment and mortality records were examined in the 2000 steer population, where no correlations with ADH1C, implant status or VA level were observed. Due to no VA × ADH1C interaction, this nutrigenetic marker-assisted management strategy is not effective at this time in commercial beef cattle feedlots, however, supplementing VA at a level as low as 25% of recommended in finishing rations would likely not result in signs of immune dysfunction.

Interaction of vitamin A supplementation level with ADH1C genotype on intramuscular fat in beef steers.

Previously, the single nucleotide polymorphism in alcohol dehydrogenase (ADH1C c.-64T>C) was shown to have an association with intramuscular fat (IMF) in the longissimus thoracis (LT) muscle when vitamin A was limited in finishing rations of beef steers. The purpose of this study was to determine the optimum vitamin A supplementation level, in combination with ADH1C genotype, to increase IMF of the LT muscle. In total, 45 TT genotype, 45 CT and 27 CC Black Angus crossbred steers were backgrounded on a commercial ration containing 3360 IU vitamin A/kg dry matter (DM). During finishing, the steers were randomly assigned to one of three vitamin A treatments at 25%, 50% and 75% of the National Research Council recommendation of 2200 IU/kg DM. Treatments were administered via an oral bolus. Carcass quality was evaluated and a sample from the LT muscle was collected for analysis of IMF. A treatment×genotype interaction (P=0.04) was observed for IMF; TT steers on the 75% treatment had higher IMF relative to CT and CC steers on the same treatment. Western blot analysis showed that TT steers had higher (P=0.02) ADH1C protein expression in hepatic tissue. Previously, TT steers exhibited increased IMF when fed limited vitamin A. In the current study, the lack of variation in IMF between treatments and genotypes at the lower vitamin A treatment levels was likely due to the majority of the steers grading Canada AAA (USDA Choice). However, the western blot data supports that TT steers are expected to have higher IMF deposition, due to an increased production of ADH1C. The interaction between ADH1C genotype and vitamin A supplementation level has the potential for use in marker-assisted management programs to target niche markets based on increased marbling.

Development of the Canadian beef reference herd for gene mapping studies.

A project to map quantitative trait loci (QTL), in beef cattle using a full-sib design was initiated using six Bos taurus breeds. Embryo transfer was used in a large scale, short timeframe experiment to develop this herd for gene mapping. Full-sib families allowed for genetic information to be followed through both the sire and the dam and for both parents to be slaughtered so that carcass quality data could also be obtained from both of them at close to typical slaughter ages. Repeatability of response to superovulation was significant among the 3 flushes per female. Response to superovulation was negatively correlated with backfat of the donor. Crossbred embryos were found to have higher survival than purebred embryos.

The impact of vitamin A restriction and ADH1C genotype on marbling in feedlot steers.

A novel SNP was discovered within the promoter region of alcohol dehydrogenase 1C (ADH1C c.-64T>C), the C allele eliminating a potential binding site for the transcription factor C/EPBα. The purpose of this study was to examine if an interaction between this SNP and vitamin A restriction had an effect on carcass characteristics in beef cattle. Following backgrounding on a ß-carotene-deficient diet, 130 steers (50 TT, 50 CT, and 30 CC) were finished for 5 mo and received either no supplemental vitamin A (unsupplemented) or 750,000 IU/mo (supplemented). A subgroup of 5 steers • genotype(-1) • treatment(-1) was randomly selected for pre- and postfinishing liver biopsies to assess vitamin A status and measure gene expression. Unsupplemented steers (Bos taurus) had significantly greater (P < 0.05) marbling scores than supplemented steers. There was a significant interaction between genotype and vitamin A supplementation on ether-extractable intramuscular fat (IMF). Within the unsupplemented treatment, TT steers had nearly 23% greater IMF than CC steers. Additionally, unsupplemented TT steers had over 24% greater IMF than supplemented TT steers. Expression of ADH1C in the liver was additive with each additional T allele, potentially due to the elimination of a possible binding site for C/EBPα. It is plausible that CC cattle have reduced ability to metabolize retinol to retinaldehyde (and subsequently retinoic acid) and that a phenotypic effect is only observed when vitamin A is limiting. Therefore, ADH1C c.-64T>C genotype, in combination with reduced vitamin A supplementation, could potentially be implemented in marker-assisted management to maximize marbling in finishing cattle.

Genetic diversity and admixture among Canadian, Mountain and Moorland and Nordic pony populations.

As part of the requirements of the Convention on Biological Diversity, Canada has been investigating the genetic diversity of its native equine and pony populations. Along with examining four indigenous Canadian equine populations (Canadian horse, Lac La Croix pony, Newfoundland pony and Sable Island population), another 10 Mountain and Moorland, three Nordic, four horse and two feral equine populations (thought to have influenced some pony breeds) were also investigated. In total, 821 individuals were genotyped at 38 microsatellite loci. Results of the analysis of molecular variance indicated that 13.3% of genetic diversity was explained by breed differences, whereas 84.6% and 2.1% of diversity came from within and among individuals, respectively. The average effective number of alleles and allelic richness was the lowest in the Eriskay (2.51 and 3.98) and Lac La Croix (2.83 and 4.01) populations, whereas it was highest in the New Forest (4.31 and 6.01) and Welsh (4.33 and 5.87) breeds, followed closely by the Newfoundland-CDN (4.23 and 5.86) population. Expected heterozygosities varied from 0.61 in the Lac La Croix to 0.74 in the Welsh and in Newfoundland. Observed heterozygosities ranged from 0.57 in the Exmoor and 0.58 in the Sable Island herd to 0.77 in the Kerry Bog and 0.76 in the New Forest breeds. Structure and admixture analyses revealed that the most likely number of clusters was 21, although some substructure was also observed when K = 16, compared with the 24 predefined populations. Information gathered from this study should be combined with other available phenotypic and pedigree data to develop, or amend, a suitable conservation strategy for all populations examined.

Single nucleotide polymorphisms in the corticotrophin-releasing hormone and pro-opiomelancortin genes are associated with growth and carcass yield in beef cattle.

A single nucleotide polymorphism (SNP) in the corticotrophin-releasing hormone gene (CRH C22G) alters the fourth amino acid in the signal sequence from proline to arginine. Two other SNPs (CRH A145G and C240G) occur in the propeptide region at residue positions 45 and 77, respectively, that result in serine/asparagine and histidine/aspartic acid substitutions respectively. These SNPs, as well as SNPs in pro-opiomelancortin (POMC), leptin (LEP) and melanocortin-4 receptor (MC4R), were evaluated for associations with average daily gain, end-of-test rib-eye area, shipping weight and hot carcass weight in a group of 256 steers using a general linear model. The CRH C22G SNP was associated with end-of-test rib-eye area (P < 0.034) and hot carcass weight (P < 0.0015). The SNP in POMC was associated with shipping weight (P < 0.0078) and hot carcass weight (P = 0.006) while it approached significance for average daily gain (P < 0.07). The SNP in MC4R approached significance for hot carcass weight (P < 0.085) while no significance was observed between the leptin SNP and the above listed traits. Because both CRH and POMC regulate appetite, potential interaction effects between these two genes were assessed. The absence of an interaction effect between CRH and POMC with hot carcass weight suggests that these genes act independently to increase carcass yield. These gene effects used singularly or together could result in an economic benefit to the beef industry.

Coelomomyces psorophorae var tasmaniensis Couch + Laird (1988) (Coelomomycetaeceae: Blastocladiales), a fungal pathogen of the mosquito Aedes australis. I: Structural changes in the outer walls of sporangia during germination.

The germination of sporangia in Coelomomyces psorophorae var tasmaniensis (C. p. tas.) is uncoordinated and thus there is a mixture of developmental stages in any given population. Continuous urografin gradients separated out the critical stages of germinating sporangia giving four bands, each band representing a consecutive stage of germination. These stages were investigated for changes in the sporangial wall using Transmission Electron Microscopy (TEM). The sporangia have a typical two-layered wall, an electron dense outer layer which can be divided into three distinct sub-layers D1, D2, and D3 and an inner electron transparent secondary wall. Stage 3 sporangia have an intact D1 layer on their outer wall. In the subsequent stages (4 & 4b) there is a progressive breakdown of this layer.

Coelomomyces psorophorae var tasmaniensis Couch + Laird (1988) (Coelomomycetaeceae: Blastocladiales), a fungal pathogen of the mosquito Aedes australis. II: Nuclear changes during meiospore formation.

The presence of synaptonemal complexes were checked in dividing chromosomes as evidence for meiotic division in germinating sporangia. Continuous urografin gradients were used to separate out the various phases of germinating sporangia, the nuclei were removed and embedded for ultrastructural studies. Meiotic inhibitors were applied to germinating sporangia to retard meiotic division to highlight the synaptonemal complexes. At an early phase of sporangial differentiation dividing nuclei developed with synaptonemal complexes. Meiotic inhibitors and stimulators may be used to control sporangial germination for an induction of a high meiospore count. This may be of crucial importance in the utilization of Coelomomyces spp. as a biological control agent of mosquito species.

DNA fingerprinting analysis of Booroola pedigrees: a search for linkage to the Booroola gene.

Seven minisatellite probes from a variety of sources were used to analyse 11 paternal half-sib families in which the Booroola gene was segregating. A total of 402 bands that showed segregation in the pedigrees were examined for linkage to the Booroola gene. None of the bands showed segregation with the Booroola gene. The most likely evidence for a linked band was produced by the HaRas HVR probe in Family 902 (θ=0.0; LOD 2.3). The conclusion, however, is that the minisatellite probes used in this study could not be used as markers for the Booroola gene. The study highlighted problems associated with the use of minisatellite probes in linkage studies in half-sib families. The complex banding patterns found on fingerprinting gels was a major source of scoring error. In a few cases both of the sire's alleles could be identified at a particular locus, but in most cases only one of the alleles could be identified. For the most part, the bands had to be treated as dominant alleles. The contribution of dam alleles to the banding pattern could only be estimated. There was an indication that minisatellite loci in sheep are clustered in particular regions of the sheep genome as the rate at which bands segregated with each other was higher than one would expect from loci randomly distributed throughout the genome.

Microsatellites from the beluga whale Delphinapterus leucas.

Fifteen microsatellites were isolated from a beluga whale Delphinapterus leucas, genomic library. The microsatellites were amplified in 100 beluga obtained from two widely separated locations. An average of 8.6 alleles per locus were detected and the average heterozygosity was 0.65 with a range of 0.27-0.86. All microsatellites were polymorphic and 13 of the genotype distributions observed were in Hardy-Weinberg equilibrium. It was possible with these microsatellites to assign correctly individual whales to their stock-of-origin 98% of the time. Microsatellites were amplified in 15 other cetaceans with these beluga-derived primers.

Microsatellites and associated repetitive elements in the sheep genome.

To determine the frequency and clustering of a variety of simple di- and trinucleotide repeats, an Artiodactyl short interspersed element (SINE), an ovine satellite repeat, and a human Alu 1 repeat were used to screen a random selection of cosmids containing inserts of ovine genomic DNA. In total, 197 individual cosmids were digested with EcoRI and the fragments separated on 0.7% agarose gels. Southern blots of these gels were then sequentially probed with (AC)7, (CT)9, and (CAC)6 oligonucleotides, and the repeats described above. The frequency at which (AC)n, (CT)n, and (CAC)n repeats were found in the cosmids indicated that they occurred at average intervals of 65 kb, 367 kb, and 213 kb respectively within the ovine genome. The Artiodactyl SINE was the most common, occurring at an average interval of 20 kb. No human Alu 1 sequences were detected. There was a significant positive association between the (AC)n and the Artiodactyl SINE. This association is quite strong as there was significant clustering of the two repeats both within cosmids and also within the EcoRI fragments of the digested genomic fragments. With the exception of the sheep satellite sequence, which occurs in tandem arrays, none of the other repeats showed significant clustering within the 41-kb (average size) cosmid inserts. The first 25 ovine microsatellites we characterized had an average polymorphic information content (PIC) of 0.65. The different microsatellite types, containing either perfect, imperfect, or compound repeats, had similar average PICs of 0.64, 0.65, and 0.66 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A missense mutation in the bovine MGF gene is associated with the roan phenotype in Belgian Blue and Shorthorn cattle.

The Roan locus is responsible for the coat coloration of Belgian Blue and Shorthorn cattle. The solid-colored and white animals are homozygotes, and the roan animals, with intermingled colored and white hairs, are heterozygous. The roan phenotype was mapped to cattle Chromosome (Chr) 5 with microsatellites, and a candidate gene was proposed (Charlier et al. Mamm Genome 7, 138, 1996). PCR primers to the exons of this candidate gene, the steel locus or mast cell growth factor (MGF) were designed. Solid-colored and white animals were sequenced. A missense mutation at 654 bp (amino acid 193, Ala --> Asp) was detected in these two groups. A PCR-RFLP was designed to this single base pair change, and 143 animals in total (Belgian Blue, Shorthorn, and various other breeds) were screened. In addition, the Canadian Beef Cattle Reference Herd (http://skyway. usask.ca/ approximately schmutz) was used to verify Mendelian inheritance of this marker with the phenotypic inheritance of roan. Our data suggest that this mutation in the bovine MGF gene is responsible for the roan phenotype.

Determination of evolutionary relationships among sheep breeds using microsatellites.

Eight ovine microsatellite loci were amplified in 40 to 50 unrelated individuals from six sheep populations representing five breeds: Romney, Border Leicester, Suffolk, Awassi, and both Australian and New Zealand Merino. For all of the microsatellite loci analyzed, there were highly significant differences in allele frequencies between samples from the different breeds. The allele frequencies generated can be used to determine the breed of an individual, given that it comes from one of the above breeds, to a high degree of accuracy. There were also some alleles that were found in only one breed, although these alleles were at such low frequencies that they are unlikely to be useful as markers for a breed. Genetic distances between breeds were obtained using Nei's formula to construct a phylogenetic tree. The tree grouped the Merino's in one branch and the Border Leicester, Suffolk, and Romney in another branch, while the Awassi, which was used as an outgroup, had its own branch. Using Nei's unbiased genetic distance formula to calculate the time of divergence of the British breeds from the Merino and the time of divergence between the Australian and the New Zealand Merino, we obtained t = 1094 and t = 227 years, respectively. Microsatellite genotyping in sheep appears to provide a useful tool for examining the evolutionary relationships between breeds.

A QTL study of cattle behavioral traits in embryo transfer families.

Two behavioral traits, temperament and habituation, were measured in 130 calves from 17 full-sib families which comprise the Canadian Beef Cattle Reference Herd. Using variance components, heritability was calculated as 0.36 for temperament and 0.46 for habituation. Genotyping of 162 microsatellites at approximately 20 cM intervals allowed the detection of six quantitative trait loci (QTL) for behavior traits on cattle chromosomes 1, 5, 9, 11, 14, 15.

Repeat sequences from complex ds DNA viruses can be used as minisatellite probes for DNA fingerprinting.

In a search for new fingerprinting probes for use with sheep, repeat sequences derived from five poxviruses, an iridovirus and a baculovirus were screened against DNA from sheep pedigrees. Probes constructed from portions of the parapox viruses, orf virus and papular stomatitis virus and the baculovirus from the alfalfa looper, Autographa californica, nuclear polyhedrosis virus all gave fingerprint patterns. Probes from three other poxviruses and an iridovirus did not give useful banding patterns.

Hot topic: an association between a leptin single nucleotide polymorphism and milk and protein yield.

Allelic variation (C to T transition that results in an Arg25Cys) in the leptin gene has been associated with increased fat deposition in beef cattle. We report that this same genetic variant is also present in dairy breeds. Body fat reserves play an important role in sustaining high milk production in early lactation, when energy intake is limited. To test for an association between the leptin single nucleotide polymorphism and milk productivity, we genotyped 416 Holstein cows and compared lactation performance data using a mixed model. Animals homozygous for the T allele produced more milk (1.5 kg/d vs. CC animals) and had higher somatic cell count linear scores, without significantly affecting milk fat or protein percent over the entire lactation. The increase in milk yield is most prominent in the first 100 d of lactation (2.44 kg/d), declining to 1.74 kg/d between 101 and 200 d in lactation. The milk yield advantage, observed in cows homozygous for the T allele, could represent a major economic advantage to dairy producers.

Microsatellite evolution in congeneric mammals: domestic and bighorn sheep.

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.