Ovine FABP4 Variation and Its Association With Flystrike Susceptibility.

Flystrike is a major cost and a welfare issue for the New Zealand sheep industry. There are several factors that can predispose sheep to flystrike, such as having fleecerot, a urine-stained breech, and "dags" (an accumulation of fecal matter in the wool of the breech). The FABP4 gene (FABP4) has been associated with variation in ovine fleecerot resistance, with a strong genetic correlation existing between fleecerot and flystrike occurrence. In this study, blood samples were collected from sheep with and without flystrike for DNA typing. PCR-SSCP analyses were used to genotype two regions of ovine FABP4. Sheep with the A 1 variant of FABP4 were found to be less likely (odds ratio 0.689, P = 0.014) to have flystrike than those without A 1. The likelihood of flystrike occurrence decreased as copy number of A 1 increased (odds ratio 0.695, P = 0.006). This suggests that FABP4 might be a candidate gene for flystrike resilience in sheep, although further research is required to verify this association.

Association between variation in faecal egg count for a natural mixed field-challenge of nematode parasites and TLR4 variation.

Research has shown that Toll-like receptor 4 (TLR4) is important in immune responses to some helminth parasites. In sheep, variation in the PAMP region of TLR4 may result in structurally and thus functionally different TLR4 molecules, and this may consequently lead to variation in the TLR4 response to parasite infections. This study involved three separate, but related sheep breeds (Merino, Polwarth and Corriedale sheep) and a total of 885 lambs from five New Zealand farms that underwent a mixed field-challenge from gastro-intestinal parasites. Faecal samples were collected at approximately 4 and 9 months of age and faecal egg counts (FECs) for Nematodirus spp. and Strongyle species determined, along with the total number of eggs per gram (EPG). Analysis of the five farms collectively revealed an association (P=0.023) between the presence of TLR4 variant *02 (mean 24 EPG) and the absence of the variant (mean 32 EPG) at 9 months of age. Conversely the presence of *03 had a significantly (P=0.047) higher mean Nematodirus spp. FEC (mean 42 EPG) compared to the absence (mean 28 EPG) at 9 months of age. More associations were revealed when the data were split according to the dominant faecal parasite species. With a predominantly Trichostrongylus spp. FEC group of lambs at 9 months of age, the presence of TLR4 variant *02 was found to have significantly (P=0.003) lower Nematodirus spp. FEC (mean 4 EPG), and also significantly (P=0.033) lower total FEC (mean 312 EPG) when compared to sheep without the variant (mean 15 EPG and 449 EPG, respectively). The presence of TLR4 variant *03 and *04 were associated or tended to be associated (P=0.010 and P=0.088, respectively) with higher Nematodirus spp. FEC (mean 25 EPG and 22 EPG, respectively) when compared to lambs without the variant (mean 10 EPG and 11 EPG, respectively). These results suggest that TLR4 variation may be affecting the immune response to gastro-intestinal parasites in sheep, although principally to Nematodirus spp. infections and not Strongyle species infections.

Two single nucleotide polymorphisms in the promoter of the ovine myostatin gene (MSTN) and their effect on growth and carcass muscle traits in New Zealand Romney sheep.

Myostatin is a negative regulator of muscle growth and development in mammals, and variation in ovine myostatin gene (MSTN) has been demonstrated to be associated with variation in the muscularity of sheep. Polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) was used to look for single nucleotide polymorphisms (SNPs) in a 304-bp amplicon from the promoter region of ovine MSTN. Sequence analyses revealed two previously identified SNPs (c.-2449G/C and c. -2379T/C) that resulted in three haplotypes (H1 (c.[-2449G; -2379C]), H2 (c.[-2449C; -2379C]) and H3 (c.[-2449G; -2379T]). The effect of these SNPs on growth and carcass traits was investigated in 357 NZ Romney lambs. General linear mixed-effect models revealed that sheep with the genotype c.-2449GC had a higher loin meat yield (p = 0.032) and proportion loin yield (p = 0.028), than those with the genotype c.-2449GG. The genotype c.-2379CC was associated with an increase in three weight traits: birthweight (p = 0.003), tailing weight (p = 0.009) and weaning weight (p = 0.028), when compared with the genotype c.-2379TC, but it was not found to have an association with growth rate. This suggests that c.-2379T/C has an effect that originates at, or before birth. Haplotype H3 was associated with a decrease in birthweight (p = 0.002), tailing weight (p = 0.003) and weaning weight (p = 0.011). Haplotype H2 was associated with increased loin yield (p = 0.012) and proportion loin yield (p = 0.002). The SNPs may have value as genetic markers for improved Romney breeding.

Variation in the bovine FABP4 gene affects milk yield and milk protein content in dairy cows.

Fatty acid binding proteins (FABPs) bind long-chain fatty acids and are involved in their intracellular transport. Of the known bovine FABP genes, FABP4 has been mapped to a region on chromosome 14 that contains quantitative trait loci for milk traits. This study investigated the association of FABP4 haplotypes with milk production traits in 719 Holstein-Friesian × Jersey cows. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of a variable region of the gene revealed three haplotypes (A, B and C). Five single nucleotide polymorphisms (SNPs) were identified: two in exon 3 and three in intron 3. A was associated (P=0.032) with increased milk protein percentage (present: 4.00 ± 0.02%; absent: 3.95 ± 0.02%) and B was associated (P=0.009) with increased milk yield (present: 23.81 ± 0.23 kg/d; absent: 23.06 ± 0.21 kg/d), but tended to be associated with a decrease in protein percentage and an increase in protein yield. Cows with genotypes AA, AB and AC produced less milk, but with a higher protein percentage than BC cows. This suggest that FABP4 affects milk yield and milk protein content, both economically important traits, and that further study of this gene is warranted.

A 57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter.

High glycine-tyrosine keratin-associated proteins (HGT-KAPs) are predominantly present in the orthocortex of wool fibres. They vary in abundance in different wools and have been implicated in regulating wool fibre properties, but little is known about the functional roles of these proteins in the fibre matrix. In this study, we used polymerase chain reaction--single-strand conformational polymorphism (PCR-SSCP) analysis to screen for variation in a gene encoding the ovine HGT-KAP6-1 protein. We identified three gene variants (A, B and C). Variants A and B were similar to each other, with only three nucleotide differences occurring downstream of the coding sequence. However, variant C had a 57-bp deletion that would notionally result in a loss of 19 amino acids in the protein. The presence of C was found to be associated with an increase in mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVFD) and prickle factor (percentage of fibres over 30 microns; PF). Sheep of genotype BC produced wool of greater MFD, FDSD and PF than sheep of genotypes AA, AB and BB. The CVFD was greater in the BC sheep than the AB sheep. The results suggest that variation in ovine KRTAP6-1 affects wool fibre diameter-associated traits and that the 57-bp deletion in this gene would lead to coarser wool with greater FDSD, CVFD and PF.

Genetic variations in the myostatin gene (MSTN) in New Zealand sheep breeds.

Myostatin, which is also known as growth and differentiation factor 8 (GDF8), acts as a negative regulator of skeletal muscle growth. Variation in the myostatin gene (MSTN) has been associated with variation in muscularity in many animals including sheep. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used to investigate MSTN in a diverse range of sheep breeds including the New Zealand (NZ) Romney, Coopworth, Corriedale, Dorper, Perendale, Suffolk, Merino, Dorset Down, Poll Dorset, Texel and other NZ cross-bred sheep. A total of 28 nucleotide substitutions were identified from nucleotide c.-1199 in the promoter region to c.*1813 (based on NCBI GenBank accession number DQ530260) and including the well-described substitution c.*1232G>A (MSTN g+6223G>A). Of these 28 substitutions, 3 were located in the promoter region, 3 in the 5'UTR, 11 in intron 1, 5 in intron 2 and 5 in the 3'UTR. One substitution in exon 1 (c.101G>A) potentially results in an amino acid substitution of glutamic acid (Glu) with glycine (Gly) at codon 34. Ten of these substitutions have not been reported previously. The genetic variation revealed in this study suggests this gene is more variable than hitherto reported and provides a foundation for future research into how this variation affects muscle and growth traits.

Variation in exon 10 of the ovine calpain 3 gene (CAPN3) and its association with meat yield in New Zealand Romney sheep.

Variation in the ovine CAPN3 gene was analysed using PCR-single strand conformational polymorphism, and its effect on growth and carcass traits was assessed in 513 New Zealand Romney lambs produced by 17 unrelated rams. Among the four allelic variants detected, the presence of variant *02 was found to be associated with an increased proportion of shoulder yield (absent: 32.6±0.01%; present: 33.4±0.03%; P=0.016), and tended to be associated with increased shoulder yield (lean meat yield of the shoulder expressed as a percentage of the hot carcass weight) (absent: 16.6±0.06%; present: 17.02±0.20%; P=0.067). No association was detected with growth traits or other carcass traits.

An association between lifespan and variation in insulin-like growth factor I receptor in sheep.

Longevity in livestock is a valuable trait. When productive animals live longer, fewer replacement animals need to be raised. However, selection for longevity is not commonly the focus of breeding programs as direct selection for long-lived breeding stock is virtually impossible until late in the reproductive life of the animal. Additionally the underlying genetic factors or genes associated with longevity are either not known, or not well understood. In humans, there is evidence that IGF 1 receptor (IGF1R) is involved in longevity. Polymorphism in the IGF1R gene has been associated with longevity in a number of species. Recently, 3 alleles of ovine IGF1R were identified, but no analysis of the effect of IGF1R variation on sheep longevity has been reported. In this study, associations between ovine IGF1R variation, longevity and fertility were investigated. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) was used to type IGF1R variation in 1,716 New Zealand sheep belonging to 6 breeds and 36 flocks. Ovine IGF1R C was associated with age when adjusting for flock (present 5.5 ± 0.2 yr, absent 5.0 ± 0.1 yr, P = 0.02). A general linear mixed effects model suggested an association (P = 0.06) between age and genotype, when correcting for flock. Pairwise comparison (least significant difference) of specific genotypes revealed the difference to be between AA (5.0 ± 0.1 yr) and AC (5.6 ± 0.2 yr, P = 0.02). A weak negative Pearson correlation between fertility and longevity traits was observed (r = -0.25, P < 0.01). The finding of an association between variation in IGF1R and lifespan in sheep may be useful in prolonging the lifespan of sheep.

A premature stop codon in the ADAMTS2 gene is likely to be responsible for dermatosparaxis in Dorper sheep.

We have used polymerase chain reaction-single-strand conformational polymorphism analysis to investigate variation in exon 2 of the ADAM metalloproteinase with thrombospondin type I motif, 2 (ADAMTS2) gene in 598 sheep, including three white Dorper lambs that had a pathology consistent with dermatosparaxis. Four sequence variants (A, B, C and D) were identified at this exon, with the lambs having the dermatosparaxis phenotype being uniquely B homozygous and their mothers being B-containing heterozygous for ADAMTS2. Analysis of the amplified exon 2 sequences revealed the B variant had a nucleotide substitution that creates a premature stop codon and would notionally abbreviate the ADAMTS2 peptide. The B variant was not found in any other breed aside from the white Dorper sheep that were studied.

Association between variation in faecal egg count for a mixed field-challenge of nematode parasites and ovine MHC-DQA2 polymorphism.

The selection of sheep that are resistant to gastrointestinal parasites and have lower faecal egg counts (FECs) has been the subject of extensive research. This has led to the speculation that the Major Histocompatibility Complex (MHC) genes could be used as markers to reduce FEC. In this study, associations between variation in ovine MHC-DQA2 and various measures of FEC recorded at two times (approximately 4 and 9 months of age) were investigated in a large group of New Zealand lambs (n=4676), derived from 185 different sire-lines, of a variety of breeds and raised on 25 separate farms. Pair-sample t-tests revealed that FEC for Nematodirus spp., Strongyle spp. and total FEC differed significantly between the two assessments. A total of twenty one DQA2 alleles or DQA2-DQA2-like haplotypes were identified, with allele/haplotype presence and frequency varying significantly between farms. For example, allele *0103 was observed on all farms, ranging in frequency from 0.2 to 60.9%, while haplotype *0101-*1601 was only present on one farm, in two lambs. A number of associations between the presence/absence of these alleles and egg counts were observed, but nearly all the allelic/haplotypic associations were age and parasite specific, suggesting that immune response is both age and challenge (parasite species mix) dependent. The exception was allele *1201 which was associated with increased total FECs at both 4 and 9 months of age; with it either being, or tending toward being, significantly associated with both increased Strongyle spp. and Nematodirus spp. counts as well. However, the observed increases in egg counts were small and ranged between 5 and 32 eggs per gram. In conclusion, we believe that the MHC plays an important role in parasite resistance, but that the MHC-nematode interaction is complex and thus the development of a single gene-marker based on the "MHC effect" is unlikely.

Ovine footrot: new approaches to an old disease.

Footrot is a bacterial disease that has substantial economic and welfare impacts in sheep and can be difficult to manage. Research is focussed on reducing the impact that footrot has on farmers and their flocks and better understanding the aetiology of the disease. Key areas of current research include, developing better vaccines, deploying tailored vaccines in a specific and targeted fashion on individual farms, analysing and developing better farm management practices to suit specific sheep farming environments, elucidating the virulence genes and bacterial population dynamics that drive footrot and using genetic testing in combination with selective breeding to produce stock that are more resilient to disease.

Rapid DNA extraction of pig ear tissues.

A single-step DNA isolation procedure from pig tissues was developed and the product used directly for polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis and sequencing. The procedure consists of proteinase K digestion of 2-10mg of fresh tissue, at 55 degrees C for 1h, followed by application of the products of digestion to filter paper. A 1.2mm-diameter punch of that paper has sufficient DNA to act as a template for PCR amplification. The quality of the genomic DNA appeared to be high as the PCR amplicons produced sharp banding patterns on both agarose gel electrophoresis and on SSCP analysis, and they could be used for DNA sequencing following cloning. The dried genomic DNA on filter paper can be kept at room temperature. The procedure is considered effective as it is simple, fast and inexpensive. It would be useful for large-scale genotyping and could be used to obtain genomic DNA from various tissues.

No evidence for a universal association between variation in faecal egg count for a mixed field-challenge of gastrointestinal parasites and the presence of the Ovar-DQA1 null haplotype in sheep.

We tested the association of the Ovar-DQA1 null haplotype with faecal egg count (FEC) in 4676 male lambs sourced from 185 different sire-lines and four breeds. For each lamb a faecal sample was collected at weaning (approx. 4 months of age) and mid-autumn (approx. 9 months of age) and each was typed for the presence of Ovar-DQA1 null. General Linear Mixed Models (GLMMs) were used to analyse the relationship between FEC and Ovar-DQA1 null, both across and within breeds. Two models were used, the first testing the effect of the presence/absence of Ovar-DQA1 null and the second testing the effect of the number of copies of Ovar-DQA1 null present in an animal's genotype (i.e. dominant/recessive or additive effects). Across the breeds, no significant effect of the presence of Ovar-DQA1 null on FEC for Nematodirus sp., Strongyle genera, or total FEC was detected at either 4 or 9 months of age using either of the models. Within breeds, significant associations were detected at 4 months in South African Meat Merinos, with the presence of Ovar-DQA1 null in an animal's genotype being associated with a lower strongyle (absent: n=23, geometric mean=135epg; present: n=10, geometric mean=67epg; P=0.025) and total FEC (absent: n=23, geometric mean=220epg; present: n=10, geometric mean=121epg; P=0.017). These associations were not detected at 9 months of age.

Polymorphisms in the ovine myostatin gene (MSTN) and their association with growth and carcass traits in New Zealand Romney sheep.

Myostatin is a regulator of myogenesis and has been implicated in the regulation of adiposity and in controlling the structure and function of tendons. Polymerase Chain Reaction Single-Stranded Conformational Polymorphism (PCR-SSCP) analysis of intron-1 was used to identify five variants (designated A-E) of the myostatin gene (MSTN). The effect of this genetic variation on growth and carcass traits was investigated in 517 Romney male lambs from 17 sire-lines, born on a South Island New Zealand farm. General linear mixed effect models revealed that the presence of allele A in a lamb's genotype was associated with decreased leg, loin and total yield of lean meat, whereas the presence of allele B was associated with increased loin yield and proportion loin yield (loin yield divided by total yield expressed as percentage). The effect of the number of allele copies present was investigated, and it was found that the absence of A, or the presence of two copies of B, was associated with increased mean leg yield, loin yield and total yield. Two copies of B were also associated with a decrease in proportion of shoulder yield, whereas two copies of A were associated with a decrease in proportion of loin yield. Associations with allele C were not detected. No associations of MSTN variation with birth weight, weaning weight, pre-weaning growth rate, draft age and hot carcass weight (H-W) were detected. These results suggest that variation in ovine MSTN is associated with meat production, but not birth weight or growth rate in New Zealand Romney sheep.

Polymorphism of the ovine beta3-adrenergic receptor gene (ADRB3) and its association with wool mean staple strength and yield.

We investigated the possibility that variation in ovine ADRB3 is associated with various wool traits, in particular mean staple strength (MSS). Polymerase chain reaction-single strand conformational polymorphism analysis of part of the ADRB3 intron was used to genotype 695 Merino lambs born on three farms in the South Island of New Zealand and which were shorn as 2-tooths. For each fleece, MSS, mean fibre diameter, mean staple length and yield were measured. The results from mixed-effects models and half-sib analyses suggest that ADRB3 alleles A and D have a negative impact on some wool traits, whereas ADRB3 alleles C and E appear to have a positive impact, with allele C potentially having a greater impact than allele E on MSS. This variation in the ADRB3 may assist in the genetic selection for increased MSS and yield in Merino sheep.

Development of a simple typing method for the ovine Toll-like receptor 4 gene.

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) from Gram-negative bacteria, as well as a number of other ligands. Genetic variation in the TLR gene has been associated with altered immune responses to pathogens and results in variation in disease susceptibility. The objective of this work was to develop a simple and rapid genotyping system for ovine TLR4 and of a sensitivity that would allow detection of allelic variation in this gene. While variation in exon 3 of the ovine TLR4 gene has been described previously, we describe here an improved genotyping method. This method could not only reveal the four alleles that have been reported previously, but also revealed a further three new alleles of this gene in a population of 1670 New Zealand sheep. This improved genotyping method will be useful in understanding innate immune responses in individual sheep and could also be a useful tool for large-scale immune studies in sheep production systems.

Association between variation in faecal egg count for a mixed field-challenge of nematode parasites and IGHA gene polymorphism.

Research has shown that variation in ovine immunoglobulin A (IgA) levels are associated with reduced faecal egg counts (FECs) in sheep hosting gastro-intestinal (GI) parasites. Variation in the constant region of the ovine IgA heavy alpha chain gene (IGHA) may result in structurally and functionally different IgA molecules and may consequently lead to variation in the IgA response to parasitisation. This study involved three sheep breeds (Merino, Polwarth and Corriedale) and a total of 2098 lambs from eight New Zealand farms that underwent a mixed field-challenge of nematode parasites. Faecal samples were taken at approximately 4 and 9 months of age and FECs for Nematodirus and Strongyle species determined along with total eggs per gram (EPG). Analysis of all eight farms collectively revealed no significant differences in FECs associated with the presence or absence of a particular IGHA allele. However, when the data was split into predominant challenge type groups, associations were detected. In 4-month-old lambs predominantly challenged by Nematodirus sp., the presence of the IGHA allele *01 was associated (P<0.05) with higher Strongyle FECs. In 9-month-old lambs predominantly challenged by Trichostrogylus sp., the presence of IGHA allele *02 was associated (P<0.006) with a higher mean total EPG at 9 months of age. These results suggest that IGHA gene variation will not be an effective gene-marker for reducing overall FEC but may be useful in defined or specific species challenges.