Assessment of some Biochemical Indices, Molecular Characterization of GYS1 Gene, and Genetic Diversity of Sudanese Camels - Camelus dromedaries

Abstract

Due to diversity of topics that has been dealt with in this thesis, therefore, each study is abstracted in a separate paragraph. Seasonal variations in circulating serum leptin (SL) profile of Sudanese camels (Camelus dromedaries) and its correlation to hump weight (HW) and body weight (BW) in both sexes under free grazing conditions was investigated. Thirty intact animals (15 of each sex) were chosen and divided into three age groups; 3-5, 6-8, and 8-above years. SL profiles, HW and BW were monitored simultaneously at eight times intervals during each summer, rainy and winter season throughout the year. Females in all age groups and during all seasons showed higher SL profiles, HW and BW gains when compared with males. Average season-wise SL profiles in both sexes and age groups indicated steadily increase during early mid rainy, peaked during late-rainy season, declined slowly over the mid and late winter season. The lowest SL profiles were during summer season. However, age-group variations in SL profiles were observed during different seasons. Same as SL, the HW and BW were low during summer season and both were higher during the mid rainy season with slight decrease in ensuing season and the following winter season. Sexual dimorphism in SL profiles in addition to seasonal variations, suggesting pineal-hypothalmic-hypophysial mediated mechanisms in dromedary. This study concludes that, serum leptin concentration in dromedary is associated with age, sex, and season as evidenced and positively correlated with hump and body weight gains during rainy season, where good pasture conditions are available. Camels have been widely acknowledged for their versatility and capabilities to cope with and to adapt harsh and stressful situations in frequently drought-stricken areas. Recently, non-invasive monitoring of hormones has XIII become the method of choice in both domestic and wildlife animals, as no restrain or blood sampling is required especially when dealing with large animals such as camels. Therefore, this study aimed to validate an 11-oxoetiocholanolone (5ß-3a-ol-11,17-dione) EIA measuring faecal cortisol metabolites (FCM). Five dromedaries (2 males and 3 females) were injected with ACTH (0.5mg/animal). Frequent blood samples were collected via permanent catheter in plain Vacutainer pre and post ACTH injection. Every faecal sample was collected after spontaneous defecation for five consecutive days (2 before and 3 after ACTH). Both plasma and faeces were stored at -20˚C, extracted, and analyzed with a respective EIA. Individual variations in both blood and faeces were observed. Baseline blood cortisol levels were 3.31±1.34 and 5.77±1.81 (ng/ml blood) in males and females, respectively. Peak concentrations were 30.98±2.76 and 33.99±2.15 (ng/ml blood) in males and females, respectively. Peak blood concentrations were reached 2 h after ACTH injection and returned to baseline levels after 2-5.5 h post injection of ACTH in both sexes. Baseline concentrations of FCM ranged from 164.78 to 489.5 (Mean±SE: 275±4.60) and from 110 to 2065.14 (Mean±SE: 706.93±18.13) ng/g faeces, while peak concentrations ranged from 286.66 to 2559.7 (Mean±SE: 1165.67±21.31) and 441.76 to 5169.12 (Mean±SE: 2281.26±32.07) ng/g faeces in males and females, respectively. Following the ACTH injection, FCM peak concentrations were reached earlier in males (24 h) than in females (36h), reflecting an increase of (930.8 %) and (731.2) above baseline levels in males and females, respectively. HPLC analysis indicates sex differences in the polarity of FCM where females showed more polar FCM than males. Conclusively, faecal cortisol metabolites can be used as tool for monitoring adrenocortical responses of dromedaries to different stressors in harsh conditions. XIV Glycogen synthase gene (GYS1) encodes the rate-limiting enzyme, glycogen synthase (GS) for glycogen synthesis of skeletal muscle. The GYS1 has been recently reported as promising candidate gene for production traits related to carcass characteristics in pigs emphasizing its powerfulness as molecular marker for genetic selection. Therefore, it is of great importance to study the structure and expression patterns of GYS1 gene in a promising animal with supreme ability to produce, reproduce, and potentiate protein shortage in frequently drought-stricken areas. Here, is the first report on the isolation of the cDNA encoding GYS1 from the Rectus femoris muscle of Sudanese camel (Camelus dromedarius) using polymerase chain reaction. The full coding region of a putative GYS1 gene of C. dromedarius (cGYS1) was amplified by reverse transcription PCR and cloned (gene bank accession number for nucleotides and amino acids sequence are FR667575 and CBV36870.1, respectively). The generated product contained an open reading frame (ORF) of approximately 2.2 kbp and deduced 737 amino acids in length, which is relatively similar to the coding region of GYS1 from other organisms. cGYS showed molecular weight and isoelectric point of 83.70 and 6.04, respectively. The deduced amino acid sequence revealed that cGYS1 shared the higher similarity with panda (99%), horse (99%), pig (99%), cattle (99%), human (97%), rhesus monkey (97%), dog (97%), guinea pig (97%), and rat (97%) suggesting a highly conservation manner of cGYS1 with those organisms. Phylogenetic analysis clustered the cGYS1 protein with Panda, horse, pig and cattle. A single nucleotide polymorphism (G/C) was detected in cGYS1 at position 2013 bp. Accordingly, restriction digestion with BsaWI enzyme was carried to reflect substituted nucleotides frequencies in different camel ecotypes in the Sudan. Nucleotide C was more frequent than G among camel ecotypes according to their geographical distribution, which was 0.65 in East and 0.73 in West. This result indicates that the substitution is more likely XV to occur as natural polymorphism. The level of expression pattern of cGYS1 in different camel tissues was examined using Real Time-PCR. cGYS1 was significantly higher in rectal muscle than in other tissues: liver (0.02-fold), kidney (0.13-fold), lung (0.05-fold), spleen (0.1-fold), adipose tissue (0.19-fold), and heart (0.62-fold). Based on different molecular approaches, this study concludes that, the cGYS1 can be further studied for regulation of muscle glycogen content and meat production traits related to the detected G/C-SNP that might be associated with meat production and carcass characteristic traits in camels. Camel classification in the Sudan is crudely based on the ethnic groups and geographical distribution with no reference of genetic base. Therefore, in this study, the genetic diversity of five major Sudanese camel ecotypes representing the western, central and eastern regions of the country was evaluated with 12 microsatellite markers. A total of 145 individuals (Butana, Darfur, Kenani, Lahawi and Rashidi-Kassala) were genotyped. The overall detected mean number of alleles (MNA) across ecotypes was 11.5±1.45. Among ecotypes, the lowest polymorphic information content (PIC) and expected heterozygosity (He) were observed in Rashidi-Kassala (0.61 and 0.69, respectively), while the highest were found in Darfur (0.68 and 0.73, respectively). F-statistics across ecotypes indicated significant global inbreeding coefficient estimate (FIT=0.057±0.027; P0.05), which was contributed to a higher but not significant within population inbreeding coefficient estimate (FIS=0.037±0.029; P0.05) and low but highly significant differentiation between ecotypes (FST=0.020±0.005; P0.05). The phylogenetic network analysis identified three closely related clusters projecting from sticky nodes: Butana and Darfur (cluster 1), Kenani and Lahawi (cluster 2) and Rashidi-Kassala (cluster 3). Consistently, structure cluster analysis exhibited all ecotypes as one admixed mosaic population. This study indicates that, XVI Sudanese camel ecotypes appear as admixture of two geographical twigs (east and west) and does not support the current classification of Sudanese dromedaries based on socio-ethno-geographical considerations.