Luận án tiến sĩ khả năng sản xuất và đa hình gen PRKAG3 của lợn lũng pù và lợn bản

1 INTRODUCTION THE RATIONALE OF THE STUDY Lung Pu and Ban (Hoa Binh Ban) pigs are native breeds which have a great potential to be exploited/utilized and developed because of their medium body size, a better productivity compared to that of other native breeds, a relatively high number of population, homogenous appearance and especially of their good adoption to harsh living conditions in high rocky mountainous areas There have been several studies on Ban and Lung Pu pigs However, these studies were only ended up with an assessment of the origin, characteristics of appearance and some production traits of these animals There has not been any study systematically evaluating genetic potentials of production traits, especially relationships between phenotypes and genotypes of meat quality traits of these breeds Due to the above mentioned reality, it is essential to carry out a research to systematically and adequately evaluate characteristics of appearance and performance, associated with genetic polymorphisms of PRKAG3 gene and examine ìnluence of this gene on the quality of meat of the native pig breeds The results of this research will contribute to effective orientations of selection, utilization of genetic resources of the breeds for enhancement of sustainability of pig production systems For all above reason, a research project on "Performance and polymorphism of PRKAG3 gene of Lung Pu and Ban pigs" was undertaken OBJECTIVES OF THE STUDY Evaluate appearance characteristics, perfomance, and influence of genetic polymorphisms of PRKAG3 gene associated with meat quality of Lung Pu and Ban pigs for breeding programs and development orientations of these two pig breeds SCIENCTIFIC AND PRACTICAL SIGNIFICANCES OF THE STUDY 3.1 Scientific significance - The study is the first scientific work systematically dealing with characterization of appearance, growth and reproduction as well as meat yield and quality of Lung Pu and Ban pigs - This is also the first scientific work dealing with genetic polymorphisms of PRKAG3 gene and its influence on some meat quality parametters (meat color) in Lung Pu and Ban pigs - Results of the study can be used as a good reference resource in research and teaching on genetic resources of indigenous pigs 3.2 Practical significance - Results of the study could be a basis for an orientation of selection to improve quality of Lung Pu and Ban pigs - Results of the study on performance could be a scientific basis for improvement of rearing process of two pig breeds in household conditions in high mountainous areas NEW FINDINGS OF THE STUDY - Full and systematic assessment of characteristics of appearance from hair coat and skin color, reproduction, growth, carcass yield and meat quality of Lung Pu and Ban pigs - Identifying PRKAG3 genetic polymorphisms including polymorphism : G52S/HphI, T30N/StyI, V199I/BsaHI, R200Q/BsrBI in the pig breeds: Lung Pu and Ban pig - Initial assessment of influences of PRKAG3 genetic polymorphisms on meat quality of Ban pig Polymorphisms of T30N/StyI and G52S/HphI affected the brightness and polymorphisms of T30N/StyI affected the yellowness of meat CHAPTER I LITERATURE REVIEW 1.1 Appearance characteristic of native pig breeds There is a variety of indigenous breeds of Vietnam, they are distributed throughout all the regions of the country with unique features It was shown that Vietnam now has about 26 native pig breeds (Ta Thi Bich Duyen et al., 2013; Nguyen Van Duc, 2012) There have been several studies on Lung Pu pig (Nguyen Van Duc, 2005; Nguyen Van Duc et al., 2008; Nguyen Van Duc et al., 2012; Trinh Quang Phong et al., 2009; Trinh Quang Phong et al., 2011) and research on Ban pig (Quach Van Thong, 2009; Vu Dinh Ton and Phan Dang Thang, 2009) 1.2 Reproductive performance of native pig breeds Most indigenous breeds have the first estrus at an early age, a low reproductive performance (5-8 piglets/litter) The number of litters/sow/year ranges from 1.1 to 1.5 However, some native pig breeds have a higher reproductive performance , for example Mong Cai pig (10 - 15 piglets/litter and 1.5-2 litters/year) (Phan Xuan Hao and Thanh Ngoc Van, 2010; Tu Quang Hien et al., 2004; Nguyen Manh Cuong et al., 2010; Ho Trung Thong et al., 2011), Lung Pu pig (Vu Ngoc Son et al., 2009; Nguyen Van Duc et al., 2008; Trinh Quang Phong et al., 2011; Trinh Quang Phong et al., 2012; Nguyen Van Duc et al., 2010) and other pig breeds (Vu Dinh Ton and Phan Dang Thang, 2009; Vu Dinh Ton et al., 2012; Quach Van Thong, 2009) 1.3 Growth, productivity and quality of the meat of native pigs In recent years, there have been many works dealing with growth, meat production and quality of meat of native pig breeds such as Nguyen Ngoc Phuc, et al., 2010; Phan Xuan Hao and Ngoc Van Thanh, 2009; Tran Thanh Van and Dinh Thu Ha, 2005; Le Dinh Cuong et al., 2004; Ho Trung Thong et al., 2011 These works have been concluding that growth, meat production of native pig breeds are not high There have also been some studies and publications on growth, meat production of Lung Pu pigs (Vu Ngoc Son et al., 2009; Nguyen Van Duc et al., 2008; Trinh Quang Phong, 2011; Nguyen Van Duc et al., 2008) and of Ban pigs (Vu Dinh Ton and Phan Dang Thang, 2009; Quach Van Thong, 2009) However, these studies have not been providing us with a sufficient information on growth and meat production of these native pigs Pig meat quality has nor been studying in the above studies Therefore, in this study, in addition to assessing reproduction, growth, meat production and meat quality of these pigs will be systematically and fully evaluated in both sexes 1.4 Genes related to pig meat quality In Vietnam, up to now there have been some studies to identify molecular markers (DNA) in animals using molecular genetic techniques to find out DNA markers associated with production traits (Le Minh Sat, 1997, Dinh Van Chinh et al., 1999; Nguyen Ngoc Tuan and Tran Thi Dan, 2005; Do Vo Anh Khoa et al., 2010) A few studies on genes related to meat quality and productivity of indigenous breeds and hybrid breeds (domestic x exotic breeds) have been published in the country (Nguyen Van Hau et al., 2000; Le Thi Thuy et al., 2004; Nguyen Van Cuong et al., 2003) PRKAG3 gene has been considering to have possible effects on meat quality of many exotic breeds (Ciobanu et al., 2001; Milan et al., 2000; Anna Grandlund et al., 2011; Meadus et al., 2002 ; Skrlep et al., 2009) and a number of Chinese indigenous pig breeds (LuSheng Huang et al., 2004) However, this gene has not been studying in any pig breed in Vietnam Therefore, polymorphism of PRKAG3 gene in two pig breeds of Lung Pu and Ban and influences of this genetic polymorphism on meat quality are questions to be answered in this study CHAPTER II MATERIALS AND METHODS 2.1 Research subjects - Evaluate the performance characteristics of 303 Lung Pu pigs, 277 Ban pigs in generations: parents and offspring Assessment of productivity of 218 litters in Lung Pu pig, 213 litters in Ban pig - Assess the growth (37 Lung Pu pigs, 38 Ban pigs), the ability for the meat producing and meat quality (30 Lung Pu pigs, 29 Ban pigs) - Identify genetic polymorphism of PRKAG3 in 150 individual Lung Pu pigs and 150 individual Ban pigs - Lung Lung Pigs were raised in Lung Pu commune, Meo Vac district, Ha Giang province Ban pigs are raised in Phu Cuong commune, Tan Lac district, Hoa Binh province - Study was carried out from 01/2012 to 06/2015 2.2 Research content 2.2.1 Identifying appearance characteristics and performance of Lung Pu and Ban pig - Characteristics of hair coat and skin color of Lung Pu and Ban pig - Reproductive performnce of Lung Pu and Ban pig - Growth of Lung Pu and Ban pig - Carcass yield and meat quality of Lung Pu and Ban pigs 2.2.2 Identifying genetic polymorphisms of PRKAG3 gene in Lung Pu and Ban pigs and effects of this gene on meat quality of Ban pig - Identify PRKAG3 gene polymorphism in Lung Pu and Ban pig - Determine possible effects of PRKAG3 gene polymorphisms on meat quality of Ban pig 2.3 Methods 2.3.1 Identifying appearance characteristics and performance of Lung Pu and Ban pig 2.3.1.1 Appearance characteristic of Lung Pu and Ban pigs Study on appearance characteristics mainly focused on evaluation of hair coat and skin color characteristics of Lung Pu and Ban pig according to criteria: Lung Pu pigs: (i) black body, (ii) black body with white-dotted points (from four elbows to the feet, forehead and tip of the tail) and (iii) black body with white rippling abdomen; Ban pigs: (i) black body, (ii) black body with white pedicures and (iii) body black with a white rippling abdominal 2.3.1.2 Reproductive performance of Lung Pu and Ban pigs - For each breed, 40 gilts and replaced boars were chosen Selected individual boar, gilt selected was not relative Average weight of selected pigs was 20 kg/head - Two experiments for two breeds were conducted Each experiment was arranged in 10 households (4 pigs/household) with similar farming conditions 40 sows were put in pair with males for rotational mating from first litter to the sixth Vietnam standards ISO 1547-2007 for nutritive requirements was used for rearing animals - Monitoring performance of experimental pigs at farms with recording books 2.3.1.3 Growth of Lung Pu and Ban pigs - Before observation, experimental pigs were chosen and identified by ear tags, weighed The experiment was repeated times, and ratio of castrated male to female was to - Raising condition: Locally available feeds were used to formulate rations according Vietnam standards ISO 1547-2007 for nutritive requirements of native pigs - Monitoring growth of pigs from months of age to month old by monthly and individually weighing and recording 2.3.1.4 - Carcass yield and meat quality of Lung Pu and Ban pigs a) Yield of meat: - Choosing pigs for slaughtering: Slaughtered pigs were selected from pigs in growth experiments based on their growth rate Ratio of male to female was to - The classical method was used for slaughtering tests, the left half of carcarss was used to separate meat, fat, bone, skin and weighed different parts according to Vietnam Standard (TCVN 38991984) b) Quality of meat: - Samples of fillet meat were taken immediately after slaughtering in position of 13th rib to and 14th rib, preserved in ice boxes and transported to the laboratory Fillet meat was cut into parts with a thickness of cm, then stored at 4°C for analysis of meat quality - pH45 and pH24 values, meat color, water loss rate of preservation and processing were determined by a method of Warner et al (1997).Toughness (softness) of meat was determined by method of Channon et al (2003) 2.3.2 Identifying genetic polymorphisms of PRKAG3 gene in Lung Pu and Ban pigs and effects of this gene on meat quality of Ban pig 2.3.2.1 Identifying genetic polymorphisms and sequencing of PRKAG3 gene - Extraction of DNA from tissue samples was done by using DNA kit of Bioneer (South Korea) - Identify PRKAG3 polymorphic gene segments was performed using PCR-RFLP: - Sequencing PRKAG3 gene was done by using Sanger method on sequencer machine ABI3130 - Analysis of PRKAG3 gene sequences was performed with a profestional software BioEdit v7.2.5 at Key Laboratory Animal Cells, NIAS 2.3.2.2 Determine possible effects of PRKAG3 gene polymorphisms on meat quality of Ban pig After identifying PRKAG3 genotype polymorphism, individual pigs carrying the typical genotype were marked, continuously monitored up to months of age and slaughtered to evaluate the relationship of each genotype with meat quality 2.4 DATA PROCESSING Data were statistically analyzed using SAS 9.0 software (2002), to calculate the statistical parameters LSM, SE, LSMeans with pair comparison pdiff Tukey corrected using Turkey method with analytical model as follows: Yijkl = μ + Bi + Lj + Nk + Ɛijkl Where: Yijkl: Obsevered values of parameter μ: Population average Bi: Effect of boar i (Lung Pu = to 4; iBAN = to 4) Lj: Effect of parity j (j = from to 6) Nk: Effect of the year k (k = from 2012 to 2015) Ɛijkl: random error CHAPTER III RESULTS AND DISCUSSION 3.1 APPEARANCE CHARACTERISTICS AND PERFORMANCE OF LUNG PU AND BAN PIGS 3.1.1 Appearance characteristic of Lung Pu and Ban pigs Lung Pu pig had prominent features such as a black body with white-dotted points The percentage of pig having this character was relatively high (65.68%) This figure for parents' generation was higher than that of the offspring generation (75.00 vs 64.09%) and the difference was not statistically significant (P [...]... pigs 3.2 PRKAG3 gene polymorphism on Lung Pu, Ban pigs and Its effects on meat quality of Ban pig 3.2.1 PRKAG3 Gene polymorphism 3.2.1.1 Specific amplification of exon 1 and exon 3 region on PRKAG3 gene Amplication of PRKAG3 gene fragment on exon 1 and exon 3 was successfully with sizes of 270 bp và 258 bp It was fuond out that only one light DNA band appeared The PRKAG3- exon1 and exon 3 gene fragments... BsrBI enzyme as reported (Figure 4) 3.2.2 Genotype and allele frequencies of PRKAG3 gene polymorphisms 3.2.2.1 Lung Pu pigs Results of allele and genotype frequency analysis in the exon 1 and PRKAG3 exon 3 polymorphisms of Lung Pu pigs were presented in Table 14 Table 11: Allele and genotype frequency in PRKAG3 gene polymorphisms of Lung Pu pigs Polymorphism Genotype frequency Allele frequency G52S/HphI... 3.2.3 Influence of PRKAG3 gen polymorphism on meat quality of Ban pigs 3.2.3.1 G52S/HphI polymorphism Water loss percentage by processing of SG genotype was higher than that of GG genotype Water loss percentage by preservation of SG genotype was lower than that of GG genotype However, no difference in water loss percentage between two genotypes was found (P>0, 05) L* values of GG genotype were higher... that of SG genotype, and differences were statistical significant (P 0.05) The toughness of Ban pig in animals with GG genotype (61.89 N) was lower than that in animals with SG genotype... only one light DNA band appeared The PRKAG3- exon1 and exon 3 gene fragments were multiplied corresponding to gene coding sequence with codes PRKAG3 AF214521.1 on www.ncbi.nlm.nih.gov gene banks 3.2.1.2 Exon 1 and exon 3 regional polymorphic of PRKAG3 gene a) The exon 1 polymorphism of PRKAG3 gene (G52S/HphI and T30N/StyI) The first polymorphisms at positions 154, which had changed nucleotide between... homozygous genotype, R allele frequency were 1.00 and 1.00, respectively Thus, corresponding genotypes existed in each polymorphism of Ban pigs in the study, specifically G52S/HphI and T30N/StyI polymorphism had 3 genotypes in the population; V199I/BsaHI polymorphisms, VV genotype was more dominant in the population than IV genotype, yet R200Q/BsrBI polymorphism II genotype have not been found and only RR genotype... genotypes At position of T30N, 2 types of allele T and N were detected corresponding to 3 genotypes of NN, TN and TT and appeared in two pig breeds studied Polymorphism at position G52S and T30N of PRKAG3 gene in Lung Pu and Ban pigs was in agreement with the work done by Sheng Huang Lu et al (2004), who found 9/21 indigenous breeds in China having polymorphism at position G52S and T30N of PRKAG3 gene... R allele corresponding to RR genotype From analysis of results, it was shown that primers used to specifically amplify and analyze the polymorphism in PRKAG3 3 gene exon 1 and exon 3 regions in Lung Pu and Ban pigs were specific, accurate and consistent with the study published in the world Figure 1: Image of PRKAG3 gene sequencing exon 1 region Figure 2: Image of PRKAG3 gene sequencing exon 3 region... of PRKAG3 gene b) PRKAG3 exon 3 gene Polymorphism (V199I/BsaHI and R200Q/BsrBI) Analyzing the V199I exon 3 gene PRKAG3 polymorphism in Lung Pu and Ban pigs, we detected 2 alleles that were V and I (Figure 15) However, only 2 genotypes of VV and IV were found The above results were corresponding to Lu-Sheng Huang et al (2004) who reported that I allele was not present in Chinese indigenous breeds and... region 21 3.2.1.3 Sequencing Results of PRKAG3 exon 1 gene sequence and analysis showed that these gene regions were amplified specifically, polymorphic points identified at A89C A154G positions were completely accurate and corresponded to points cut by HphI and StyI enzymes (Figure 3) The results of sequence and analyze PRKAG3 exon 3 gene region also showed that this gene segment was multiplied specifically