This thesis contains no text, images or tables copied and pasted from the internet unless specifically stated, and the source is described in detail within the thesis and in the References sections. Thanks to Dr. Mervyn Beukes whose generosity to his students with his molecular knowledge and laboratory techniques helped us save a lot of time in the laboratory. 29 Table 2.2 Classification Characteristics of beef carcasses according to SAMIC 31 Table 2.3 Primers for amplification of lep exon 2.
Magnesium chloride
ABSTRACT
Cattle breeds
There are three main types of cattle, Bos taurus (humpedless), commonly referred to as taurine, Bos indicus (humped), which is known as Zebu, and the hybrid Bos taurus africanus, which includes the Sanga cattle breeds (Loftus et al., 1994) ) . The most notable dairy breeds include the Holstein-Friesian, Ayrshire and Jersey, while major beef breeds include the Angus, Hereford, Charlois and Brahman, although many composite breeds have been developed in pursuit of specific economic traits valued by different groups throughout the country (Porter, 1991; .Casas et al., 2000). In South Africa, indigenous and locally developed breeds such as the Nguni, Tuli, Afrikaner, Bonsmara, Drakensburger, Huguenot and Sanganer are considered primarily beef breeds (Porter, 1991; Scholtz et al., 1999a; Ramsay et al., 2000).
Beef industry in South Africa
- Introduction
- Beef breeds
- Economically important traits in the beef industry
- Beef breeding
The Nguni have inherent adaptability to the harsh climate and the various agricultural systems practiced in South Africa (Scholtz et al., 1999a). The Nguni are perceived as a low maintenance (minimal care) breed with high performance, making them highly profitable animals (Ramsay et al., 2000). These values provide an indication of how the traits will respond simultaneously to selection (Golden et al., 2008).
Molecular markers in beef breeding
- Marker assisted selection (MAS) in beef cattle
The consequence of the linked marker is that there is no guarantee of identifying the exact QTL (Kinghorn et al., 2000). No knowledge of the DNA sequence of the target gene is required and the primers bind randomly to the DNA sequence (Williams et al., 1990). Although the effects of QTL on all relevant traits must be somewhat known before embarking on breeding strategies (Kinghorn et al., 2000).
Role of leptin in the beef industry
- Introduction
- Leptin locus as a molecular marker in the beef industry
Two cysteine residues (Cys 146 and Cys 96) form an intrachain disulfide bridge between the C-terminus, COOH, and the beginning of the CD structure of the protein (Zhang et al., 1997). In addition, Buchanan et al. 2001) identified a cytosine (C) to thymine (T) transition that encoded an amino acid change from an arginine to a cysteine in exon two of the leptin gene. Further investigation and application of the SNP (E2FB) has extended to positive associations with feed intake, intramuscular fat, yield and milk composition (Buchanan et al., 2002).
Investigation 1: Assessment of phenotypic variation
- Introduction
- Methods
The value-based marketing system of the South African beef industry is partly from carcasses without excess fat covering and carcasses are graded according to the legislation set out by the government organization of SAMIC (Perry and Fox, 1997). A body composition score (BCS) was assigned to each animal on the first day of the feedlot period, while live weight was recorded on day 1 and day 113. The BCS was determined according to the guidelines specified by Webster (1987), the tail head area and loin area of the animal were assigned a grade according to conformation and fatness levels.
Investigation 2: Assessment of genetic variation at the lep locus
- Introduction
- Methods: DNA source and isolation
- Methods: Lep gene amplification
- Primer set of lep gene exon 2
- Amplification of lep gene exon 2
- Verification of amplification product
- Methods: Lep gene restriction
- Methods: Determination of lep alleles and genotypes
- Methods: Population analysis of the lep locus
The tube was then vortexed until the bottom of the tube no longer contained debris. The tube was then gently shaken for 15 minutes to avoid DNA fragmentation. 7 l of the sample and the marker were carefully loaded with a micropipette into separate wells in the polyacrylamide gel.
Investigation 3 and 4: Assessment of associations between lep genotypes with phenotype and a comparative analysis between Nguni
- Introduction
- Methods
Where 𝑌𝑖𝑗𝑘𝑙𝑚𝑛𝑜 is the trait measured for an individual; 𝑢= overall mean for the trait; 𝑏1𝐿𝑖= regression coefficient 𝑏1 of the covariate initial live weight 𝐿𝑖 of the ith animal; 𝐵𝑗= breed where j is Nguni or Hereford; 𝑆𝑘= fixed effects of gender, where k denotes male or female; 𝐴𝑙= age where l indicates age 1, 2 or 3 years; 𝐺𝑚= genotype where m represents the genotypes CC, CT or TT; 𝑇𝑛= where 𝑛 indicates whether the individual is hormone-treated or not 𝐵 × 𝐺 𝑗𝑚= interaction between race and genotype; 𝐵 × 𝐺 × 𝑇 𝑗𝑚𝑛= interaction between race, genotype and treatment; 𝐵 × 𝑇 × 𝑆 × 𝐴 × 𝐺 𝑗𝑘𝑙𝑚𝑛 = interaction between race, sex, age and genotype; and 𝑒𝑜= environment where o represents the feedlot in Dundee or Cato ridge. Subsequently, the phenotypic response variables were individually assessed using a t-test for associations for the different genotypes, race, age and sex using the program SPSS version 11.5.1 (LEAD Technologies Inc., 2002) to obtain significance levels and correlation estimates . The nature of the relationship between the genotypes (CC, CT and TT) and the phenotypic response variables for each breed, including the fixed effects of age, sex, breed and treatment, was illustrated using an ordination graph constructed with the program Canodraw (ter Braak and Smilauer, 1998).
The association measure is expressed as an eigenvalue that demonstrates the strength of the ordination axes and quantifies the importance of the variables (Lepš and Smilauer, 1999; Okland, 1999). When different treatment groups of a variable such as bulls and heifers of the variable sex appear close to each other in the graph, it indicates that the differences between the treatment groups are small. Response variables are described in terms of the length of the arrow as well as the angle of the arrow and the ordination axis.
The longer the arrow and the smaller the angle, the stronger the association with the independent variable. RDA analysis of the associations of genotype with phenotypic response variables for the Hereford population was subdivided according to the availability of phenotypic measurements. A separate RDA analysis for breed differences compared the untreated Nguni steer group with the Hereford steers to eliminate the effects of treatment and sex.
RDA of the two breeds was first performed using selected measures of phenotypic response.
RESULTS
- Investigation 1: Assessment of phenotypic variation
- Introduction
- Investigation 2: Assessment of genetic variation at the lep locus
- Lep gene amplification and restriction
- Population analysis of the lep locus
- Investigation 3 and 4: Assessment of associations between lep genotypes and phenotypes and a comparative analysis between
- General Linear model
- Redundancy analysis
Therefore, all analyzes of Nguni data had to take treatment into account to avoid skewing results. Furthermore, no significant differences in the variation of the BCS, FAT and CFN response variables could be explained by hormone treatment. The size of the amplified product was then checked by electrophoresis in a 20% polyacrylamide gel (Figure 3.3 right).
The presence of the T nucleotide would not result in any restriction of the amplification product (94 bp). Both the T and C alleles of the SNP were present in both the Nguni and Hereford populations, as well as in the various Nguni subgroups. All three SNP genotypes, on the other hand, were present only in the Ox+ subgroup of the Nguni population (Table 3.5).
62 Trends in the distribution of TT, CT and CC genotypes for the Nguni and Hereford populations are shown by histograms in Figure 3.6. To gain a more detailed understanding of the nature of the relationships between the Nguni phenotypic response variables and the independent variables, an ordination plot was constructed (Figure 3.7). A detailed understanding of the relationships between phenotypic response variables and covariates was obtained by constructing an ordination graph (Figure 3.10).
The results of the RDA were generated in the form of a Monte Carlo test which established a highly significant relationship between breed and phenotypic response variables (Table 3.12).
DISCUSSION
Previous research also indicated a possible association of the T-allele (missense mutation) with fatter carcasses and the C-allele (normal) with leaner carcasses (Buchanan et al., 2002). This conflicts with the evidence of an association of a SNP in the lep exon 2 as identified by Buchanan et al. 2002), with carcass fat content and various carcass characteristics such as intramuscular fat, back fat and subcutaneous fat as well as carcass weight in Hereford cattle. 2005) also identified an association of the lep genotypes with fat, lean yield and intramuscular fat in crossbred cattle. The TT and CT genotypes showed significantly higher WG than CC individuals which was expected due to the skewed nature of the genotypic frequency whereby only one CC individual was present in the population.
Interestingly, the P value obtained for the Hereford data was much lower than that of the Nguni, suggesting that the two breeds are genetically different at the lep locus. Therefore, this research addresses the relevance of the lep SNP as a marker in the South African beef industry. This expected result was due to the larger frames of the older animals, although research has also shown that younger individuals produce significantly more tender meat compared to their older counterparts (Morgan et al., 2002.).
A comparison of the phenotypic response variables Nguni and Hereford by GLM yielded significant results for ILW, SLW, WG. A comparison between the overall phenotypic performance of the Nguni with the Hereford population revealed that the Hereford had significantly greater feedlotting capacity due to significantly higher WG values and SLW, WCM and CCM, which could be attributed to genetic differences between the two varieties, as confirmed by previous studies. study in Hereford (Dean et al., 1976). Thus, it appears that the native Nguni does not compare well with the exotic Hereford in the commercial AAA beef grading system, where carcass weight largely determines the value of the product.
Although the carcass characteristics and feedlot performance data of the Nguni were not favorable compared to the Hereford in this study, it is however important to note that Strydom et al. 2000) found that native beef cattle breeds were equivalent to the exotic breeds in terms of feedlot performance.
Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels. Single nucleotide polymorphisms in the corticotrophin-releasing hormone and pro-opiomelancortin genes are associated with growth and carcass yield in beef cattle. Effects of the signal transducer and activator of transcription 1 (STAT1) gene on milk production traits in Holstein dairy cattle.
Comparison of fatty acid composition of subcutaneous adipose tissue from mature Brahman and Hereford cows. A novel mutation in the coding region of the bovine leptin gene associated with food intake. Association of a single nucleotide polymorphism in the bovine leptin gene with feed intake, growth, feed efficiency, feed growth, feeding behaviour, carcass quality and body composition.
R863 in Government Gazette of the Republic of South Africa 1 September 2006 - http://www.sa-eshop.com/SAMIC- Classification-ENG1.pdf accessed. Association of single nucleotide polymorphisms in the leptin gene with carcass and meat quality traits in beef cattle. Association of a single nucleotide polymorphism in the calpastatin gene with carcass and meat quality traits in beef cattle.
The role of bovine growth hormone receptor genes in milk, fat and protein production in Finnish Ayrshire cattle.
APPENDIX A: LABORATORY REAGENTS AND CALCULATIONS
NaCl and Tris-HCl were added to approximately 80 ml d.H2O and adjusted to pH 8 with NaOH, before the addition of EDTA. 106 Adjusted to pH 8 with acetic acid. didn't add much d.H2O before adjusting the pH - a lot of acetic acid was needed to reach pH 8).
APPENDIX B: SAMPLE DATA Diet Programme
Sample Data
Tag ID/Individual ID (from jugular) Sex: 1-Ox, 2-Heifer Age Genotype BCS Starting Live Weight (Kg Slaughter Weight (Kg) Weight Gain-Feed Plot Response Fatness Conformation Warm Carcass Weight Cold Carcass Weight.
APPENDIX C: STATISTICAL ANALYSIS
Sequences results
Race slw LSMEAN wg LSMEAN Hereford Non-est Non-est Nguni Non-est Jo-est. Race yndyrë LSMEAN cfn LSMEAN wcm LSMEAN ccm LSMEAN Hereford Non-est Jo-est Non-est Nguni Non-est Jo-est Jo-est Jo-est.
