Tóm tắt Luận án The association between polymorphismsof some cadidate genes and growth performance, backfat thickness and intramuscular fat traits in duroc pigs

Nội dung text: Tóm tắt Luận án The association between polymorphismsof some cadidate genes and growth performance, backfat thickness and intramuscular fat traits in duroc pigs

1. MINISTRY OF EDUCATION AND MINISTRY OF AGRICULTURE AND TRAINING RURAL DEVELOPMENT NATIONAL INSTITUTE OF ANIMAL SCIENCE HOANG THI THUY THE ASSOCIATION BETWEEN POLYMORPHISMSOF SOME CADIDATE GENES AND GROWTH PERFORMANCE, BACKFAT THICKNESS AND INTRAMUSCULAR FAT TRAITS IN DUROC PIGS SUMMARY OF THE PhD THESIS Majors: Animal Genetics and Breeding Code: 9. 62. 01. 08 Hanoi - 2021
2. Research completed at:National Institute of Animal Science VIỆN CHĂN NUễI Scientific supervisors: 1- Ph.D Pham Doan Lan 2- Ph.D Doan Van Soan 1. TS. Phạm Doón Lõn 2. TS. Đoàn Văn Soạn The first reviewer: Assoc. Prof. Ph.D. Dong Van Quyen The second reviewer: Assoc. Prof. Ph.D. Phan Xuan Hao The third reviewer:Ph.D. Nguyen Van Hau This thesis will be defended at the Institute-level Phd Thesis Evaluation Council, which will be met at National Institute of Animal Science, Thuy Phuong, Bac Tu Liem, Hanoi. At, 2021 Thesis can be found at the library: 1. Library of National Institute of Animal Science 2. National Library of Vietnam 2
3. LIST OF SCIENTIFIC PUBLICATIONS RELATED TO THE THESIS 1. Hoang Thi Thuy, Giang Thi Thanh Nhan, Pham Thi Phuong Mai, Tran Thi Thu Thuy, Le Quang Nam, Doan Phuong Thuy, Nguyen Van Hung, Tran Xuan Manh, Doan Van Soan and Pham Doan Lan. 2019. Associations of some candidate genes polymorphisms with growth traits in Duroc pig. Livestock Research for Rural Development, Volume 31(10), October. 2. Hoang Thi Thuy, Giang Thi Thanh Nhan, Pham Thi Phuong Mai, Tran Thi Thu Thuy, Le Quang Nam, Doan Phuong Thuy, Nguyen Van Hung, Tran Xuan Manh, Doan Van Soan and Pham Doan Lan. 2021. The association between polymorphisms of some candidate genes and the growth and backfat thickness of Duroc pig over two generations. Journal of Science and Technology of Livestock. Number 264: 1 - 7. 3. Hoang Thi Thuy, Pham Thu Thao, Giang Thi Thanh Nhan, Nguyen Van Hung, Tran Xuan Manh, Doan Van Soan and Pham Doan Lan. 2021. Polymorphisms of candidate genes and their association with intramuscular fat in Duroc Pig. Journal of Livestock Science and Technology, Institute of Livestock Production. February issue. Vol 120: 90 - 98. 3
4. PREFACE 1. The necessary requirements of the thesis In recent years, the rapid development of molecular genetics hashelped the selection of livestock breedsfaster, more accurate and more efficient. Many candidate genes related to growth and meat quality had been studied and proposed to be used for selection programs supported by molecular markers such as MC4R, PIT1, GH, LEP, PIK3C3, FABP3, ADRB3, PLIN2, ACSL4genes.However, the relationship between gene polymorphisms and traits depended on the characteristics or genetic nature of each pig population at each production facility. Therefore, to apply genes to support selection of livestock breeds for each desired trait, it was necessary to conduct research to evaluate the association of candidate genes on the population which was selected. Duroc pigs were one of the foreign pig breeds that had the ability to gain weight quickly, good meat quality (soft meat due to fat mixed with lean) and high lean percentage. Therefore, in Vietnam, Duroc pigs were used in lean herd programs, which partly improved the productivity and quality of meat for the pork industry. On the other hand, many studies showed that the weight gain in Duroc pigs in Vietnam was not superior to the weight gain in Duroc pigs of some developed countries. Therefore, how to improve the ability to increase weight, backfat thickness and the quality of meat to promote the pork industry was becoming an important research direction. In order to had a scientific basis for the application of genetic markers to support the selection and improvement the ability to increase weight and meat quality of Duroc pigs fed at Dabaco Nuclear Pig Breeding Co., Ltd., I researched the topic"The association between polymorphisms of some candidate genes and growth performance, backfat thickness and intramuscular fat traits in Duroc pigs". 2. The aim of the thesis research Determine the polymorphisms of MC4R, PIT1, GH, LEP, PIK3C3 genes and their association with the traits of growth performance, backfat thickness, and reproductive performance in Duroc pigs. Determine the polymorphism of ADRB3, ACSL4, FABP3, PLIN2 genes and the association with the intramuscular fatin Duroc pigs. Select the Duroc pig line in the direction of growth performance using the support from genotype information. 3. Research contents Content 1: Studying genetic polymorphisms and association of MC4R, PIT1, GH, LEP and PIK3C3 gene polymorphisms with the traits of growth performance, backfat thickness, and reproductive performance in Duroc pigs. 1
5. Content 2: Studying genetic polymorphisms and the association of ADRB3, ACSL4, FABP3 and PLIN2 gene polymorphisms with the trait of intramuscular fat. Content 3: Selecting Duroc pigs in the direction of growth performancebased on genotype information. 4. Science and practice meaning of the thesis The thesis provided information on genotypic frequency, allele frequency and the association with growth performance, backfat thickness, reproductive performance, and intramuscular fat of some candidate genes as MC4R, PIT1, GH, LEP, PIK3C3, ADRB3, ACSL4, FABP3, PLIN2 on Duroc pigs fed at Dabaco Nuclear Pig Breeding Co., Ltd. The thesisprovided a scientific basis for the use of some candidate genes to support the selection of Duroc pigs which had highgrowth performance, backfat thickness,and intramuscular fat at Dabaco Nuclear Breeding Pig Co., Ltd. Articles published in domestic and foreign scientific journals were valuable references in scientific research and training. 5. Thenew contribution of the thesis The thesis was a systematic study including: polymorphism analysis of candidate genes MC4R, PIT1, GH, LEP, PIK3C3, ADRB3, ACSL4, FABP3, PLIN2; Evaluating the association between polymorphisms of these genes with growth performance, backfat thickness, and intramuscular fat; Application to select Duroc pigs with high growth performance based on candidate genes information at Dabaco Nuclear Pig Breeding Co., Ltd. Provide a scientific basis for orienting the use of molecular markers assisted selection to improve productivity and meat quality in breeding for Duroc pig breed, thereby shortening the selection time and improving the efficiency of livestock production, which met the requirements of high-quality and productive pig production in our country. Chapter 1. OVERVIEW With the development of molecular biotechnology, the research on DNA molecular markers had made rapid progress. DNA molecular markers were widely applied in genetic polymorphism studies, which relied on the characteristics of the DNA molecule (diversity, stability and specificity for individuals and species) to apply to breeding, evolutionary research and taxonomy.These methods were superior to traditional phenotype-based selection methods, in particular in reducing selection time and selecting for traits that had low heritability or were difficult to access phenotype or very expensive to judge by phenotype. Many candidate genes related to growth and meat quality had been studied and proposed to be used for selection programs supported by molecular markers (MAS) such as: PIT1 gene (Feng et al., 2012; Daga et al., 2012; Kim et al., 2014; Al-Khuzai et al., 2018).The MC4R gene encoded a protein which was a membrane-bound receptor. This receptor played an important role in controlling food intake, body mass and maintaining stability intracellular energy.The MC4R gene was associated with increased weight and back fat thickening (Davoli 2
6. et al., 2012; Hirose et al., 2014). The GH gene was related to the traits of carcass and growth (Bižienė et al., 2011; Lyubov et al., 2017). The LEP gene was important to the food intake control and energy balance.The LEP gene was associated with weight gain (Tempfli et al., 2015). The PIK3C3 gene was related with weight gain during the growing–finishingperiod (Hirose et al., 2011). FABP3, ADRB3, PLIN2 and ACSL4 genes which was connected to meat quality traits had the potential to be developed as markers of selection for high-fat pigs (Davoli et al., 2011; Han et al., 2012; Chen et al., 2014; Xue et al., 2015). Duroc pigs were imported from Canada and Taiwan by Dabaco Nuclear Pig Breeding Co., Ltd. from 2014 - 2018, fed in Tan Chi commune, Tien Du district, Bac Ninh province. Mature boars had a weight of 320 - 350 kg. Mature sows weigh from 250 to 280 kg. Weight gain was from 750 - 800 g per day, backfat thickness was 10 - 12 mm. Weight gain of Duroc pigs fed in Japan reached 873.6 g/day (Suzuki et al., 2005), fed in Spain reached 861 g/day (Rauw et al., 2006).DanFed Company (2014) reported that in Denmark, Duroc boars fed at the productivity control station had an average daily gain of 1,140 g/day, respectively. Thus, the weight gain of Duroc pigs from Canada and Taiwan was not superior to Duroc pigs of other origins and especially much lower than that of some developed countries. Therefore, studying how to select the traits of growth performance, backfat thickness, and intramuscular fat for Duroc pigs was an urgent requirement. Chapter 2. OBJECTS AND SCOPE OF THE THESIS RESEARCH 2.1. Objects Duroc pigs were fed at Dabaco Nuclear Pig Breeding Co., Ltd., originating from Taiwan and Canada. 2.2. Time and location Tine:from December 2016 to December 2020. Location: +Dabaco Nuclear Pig Breeding Co, Ltd; Tan Chi commune, Tien Du district, Bac Ninh province. + Key Laboratory of Animal Cell Technology, National Institute of Animal Science; Thuy Phuong ward, Bac Tu Liem district, Hanoi city. 2.3. The scope of the thesis research 2.3.1.The evaluation of the growth performance of Duroc pigs Evaluatethe growth performance of 500 Duroc gilts. Feeding mode and disease prevention: follow the breeding process of Dabaco Company. Data collection Weigh each individual at the beginning and end of the experiment using the Mettler Toledo electronic scale (China). Growth performance (g/day) was calculated based on the final body weight of each individual and the number of feeding days. Backfat thickness was measured by the Exago ultrasound machine with an Aloka SSD 500v transducer at the base of the last rib which was 6.5 cm from the vertebral line 3
7. on each individual at the same time of the end-weighing method described in the study of Youssao et al., 2002. Data processing All analyzes were processed by Minitab 16 software. 2.3.2. Methods for studying candidate gene polymorphisms + Study on polymorphism of candidate genes with weight gain and backfat thickness (MC4R, PIT1, GH, LEP and PIK3C3) was carried out on 02 generations: Generation 1 with 500 Duroc gilts (362 and 138 males); Generation 2 with 188 Duroc gilts (133 females and 55 males). + The polymorphism study of candidate genes for intramuscular fat (ADRB3, ACSL4, FABP3 and PLIN2) was performed on 200 Duroc pigs including 118 males and 82 females generated from 23 males and 69 females. + Taking samples Use specialized pliers to cut 2-3 cm of the tail of each pig when the pig was 4 days old. The tail samples were transferred into 1.5 ml tubes containing 90o ethanol solution. Samples were stored at -20°C before DNA extraction. + DNA Extraction From each tail tissue sample, DNA was extracted using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific). + PCR reaction A PCR reaction was prepared with a total volume of 25 àl consisting of 12.5 àM DreamTaq PCR Master Mix 2X (Thermo Fisher Scientific), 0.4 àM each primer and 50 ng DNA. + Detection and analysis of PCR products by Electrophoresis PCR products were checked for quality and size by electrophoresis on 2% agarose gel at 100V for about 30 minutes. Gel electrophoresis was observed under UV light by the fluorescence ethidium bromide. + Sequencing of genes PCR products of candidate genes were cleaned according to the procedure of the PureLinkđ PCR Purification Kit (Invitrogen). The sequencing process was carried out in 3 steps: performing the sequencing reaction, cleaning up after the reaction and sequencing on an ABI 3130 machine of AB (Applied Biosystem). 2.3.3.Determining the association of MC4R, PIT1, GH, LEP, PIK3C3 gene polymorphisms with average daily gain, and backfat thickness Data were handled by Minitab 16 software. General linear model GLM was used to evaluate the association between polymorphisms of genes PIT1, MC4R, GH, LEP, PIK3C3 with TKL and DML with the model: Yijk = à + Gi + SEj + G*SEij + Sk + eijk With: Yijk was growth performance or backfat thickness; 4
8. à was the population mean; Gi was the effect of genotype i of each gene (genotype i = GH: genotype AA/GG/AG; LEP: genotype TT/CT; PIK3C3: genotype TT/CC/CT; MC4R: AA/GG/ AG; PIT1: AA/AB/BB); SEj was the effect of sex j (j = male and female); G * SEij was the interaction effect between genotype and sex; Sk was the influence of the sire; eijk was the random error. Compare the confidence level between the means using Least Square Mean – LSM with Tukey comparison. 2.3.4.Determining the association of MC4R, PIT1, GH, LEP gene polymorphisms with eproductive performance Research subjects are: 104 sows; number of litters (445 litters), number of fathers (27 heads) and mothers (73 heads) of sows; The number of litters was shown in Table 2.1. Table 2.1. Parities of 104 Duroc sows Parities 1 2 3 4 5 6 Litters 104 85 81 69 56 50 General linear model GLM was used to evaluate the association between polymorphisms of PIT1, MC4R, GH, LEP genes with reproductive performance: Yij = à + Gi + eij Yij was the phenotypic value of the trait; à was the population mean; Gi was the genotype effect of each gene (genotype i = GH: genotype AA/GG/AG; LEP: genotype TT/CT; MC4R: AA/GG/AG; PIT1: AA/AB/BB); eij was the random error. Compare the confidence level between the means using Least Square Mean – LSM using Tukey comparison. 2.3.5. Association between ADRB3, ACSL4, FABP3 and PLIN2 gene polymorphisms with intramuscular fat The study was carried out on 200 Duroc pigs including 118 males and 82 females generated from 23 males and 69 females (follow-up period over a generation). The loin intramuscular fat of 200 Duroc pigs was measured using an Exago ultrasound machine and an Aloka SSD 500v transducer at the end of the performance test. The ultrasound machine with a flat transducer 12 cm long with a frequency of 3.5 MHz could scan 12.5 cm deep to capture images. The transducer was placed vertically, parallel and about 6-7 cm from the center of the animal's spine at the 10th rib position. From the images obtained by ultrasound, data on backfat thickness could be measured directly on the screen of the ultrasound machine or transferred to a computer and processed by Biosoft Toolbox II for Swine software of Biotronics.In Company. As for the intramuscular fat, it could only be measured through Biosoft software when the image data from the ultrasound machine was 5
9. transferred to the computer. Each researched individual was measured and recorded at least 5 images, corresponding to 5 repeated measurements. Then, each image (repeat) would be processed to give parameters of backfat thickness and intramuscular fat. Arithmetic average results of 5 repeated measurements would be used to evaluate and compare these criteria between researched individuals. General linear model GLM was used to evaluate the association between FABP3, ADRB3, PLIN2, ACSL4 polymorphisms with intramuscular fataccording to the model: Yijk = à + Gi + Sej + Gi*Sj + Sk + eijk With: Yijk: intramuscular fatwith genotype i and sex j; à: the population mean; Gi: fixed effect of genotype i (i=3, corresponding to 3 genotypes); Sej: fixed effect of the jth sex (j=2, male or female respectively); Gi*Sj: cumulative fixed effect of genotype i and sex j; Sk: influence of the sire; eijk: random error. Compare the confidence level between the means using Least Square Mean – LSM using Tukey comparison. 2.3.6. Selection of Duroc pigs for weight gain based on genotype Step 1: Select Duroc males (20) and females (100) with genotype homozygous for both MC4R (AA) and PIT1 (AA) genes or homozygous for 1 gene and heterozygous for 1 gene from 1000 females and 400 gilt boars. Conduct F0 generation. Bước 2: Select 60 individuals (50 females + 10 males) carrying both MC4R, (AA) and PIT1 (AA) genotypes with high growth performance from litters of F0 generation to monitor the growth performance in F1 generation. Pair to create F2 generation. Bước 3.Select sows and boars with high growth performance to access the performance in F2 generation. * Research targets: - Pre-test weight (kg); - Post-test weight (kg); - Growth performance (g per day); - Backfat thickness (mm). * The method of studying the growth performance of Duroc pigs in F1 and F2 which had both MC4R (AA) and PIT (AA) genotypes were performed as in section 2.3.1. CHAPTER 3. RESULTS AND DISCUSSION 3.1. GROWTH PERFORMANCE OF DUROC PIGS Growth performance of 500 Duroc gilts during the period of individual performance testing was presented in Table 3.1. 6
10. Table3.1. Results of Duroc pig growth performance monitoring Traits Mean ± SE CV (%) Birth weight/piglet (kg) 1.55 ± 0.01 16.91 Weaning weight/ piglet (kg) 6.68 ± 0.06 19.34 Pre-test weight/pig (kg) 31.67 ± 0.14 10.16 Post-test weight/pig (kg) 94.71 ± 0.34 8.14 Age to start testing (days) 71.52 ± 0.13 4.05 Age to finish testing (days) 149.29 ± 0.29 4.17 Number of weaning days (days) 23.39 ± 0.09 9.46 The number of test day(days) 77.99 ± 0.28 8.12 Growth performance (g per day) 809.04 ± 4.12 11.39 Backfat thickness (mm) 12.01 ± 0.08 14.41 Growth performance of 500 Duroc gilts during the individual performance testing period showed that: Duroc gilts had birth weight/piglet, weaning weight/piglet of 1.55 kg and 6.68 kg, respectively. Age to start testing was 71.52 days; age to finish testing was 149.29 days; the number of weaning days was 23.39 days; the number of test days was 77.99 days; pre-test weight/pig was 31.67 kg; Post-test weight/pig was 94.71 kg,growth performance was 809.04 g/day, backfat thickness was 12.01 mm. 3.2. GENOLOGY POLYNOLOGIES OF MC4R, PIT1, GH, LEP AND PIK3C3 3.2.1.Concentration and purity of the DNA sample The tail tissue samples of the Duroc pigswere successfully isolated. The electrophoresis image showed that the DNA was concentrated in a clear, bright, unbroken band. After measuring on Nano drop 2000, the DNA samples had high purity with the ratio A260/280 in the range of 1.79 - 2.03 and the total DNA concentration fluctuated in the range of 70-150 àg/àl. 3.2.2. Polymorphism of MC4R, PIT1, GH, LEP and PIK3C3gene segments With specifically designed primer pairs and normalized PCR reaction conditions. DNA fragments containing polymorphisms on the studied genes (MC4R, PIT1, GH, LEP and PIK3C3) were specifically cloned. PCR products of candidate genes were cut with specific enzymes. The results show that: The MC4R gene was cutby the TaqI enzyme resulting in three different genotypes (AA, AG and GG). Genotype AA had a unique band of 226 bp in size; genotype AG had 3 bands corresponding to size 226 bp, 156 bp and 70 bp; Genotype GG had2 bands with sizes 156 and 70 bp. The results of determining genotype frequencies, allele frequencies of MC4R gene polymorphisms in Duroc pig population showed thatheterozygousAG genotypes predominate 7
11. in the 1st and 2nd generation with frequencies of 0.51 and 0.48,correspondingly. The A and G allele frequencies were 0.41 and 0.59, respectively, in both generations. Figure 3.1. Genotyping at MC4Rpolymorphic site by TaqI M: Standard DNA scale 100 bp The sequencing of MC4R gene polymorphisms was shown in Figure 3.2. GenotypeAG GenotypeAA GenotypeGG Figure 3.2. Sequencing results of MC4R gene polymorphisms 8
12. For the PIT1 gene, the product was cut by RasI enzyme to create three different genotypes AA, AB and BB. Genotype AA had 4 bands with sizes 774 bp, 710 bp, 153 bp and 108 bp, respectively; genotype AB had 6 bands corresponding to size 774 bp, 710 bp, 388 bp, 322 bp, 153 bp and 108 bp; BB genotype had 5 bands with sizes 774 bp, 388 bp, 322 bp, 153 bp and 108 bp, respectively. The results of determining genotype and allele frequencies of PIT1 gene polymorphism in Duroc pig population showed the highest frequency of heterozygous AB genotypes in both generations, in the 1st and 2nd generation, 0.40; 0.41, respectively. The second highest frequency was genotype AA (0.30) in the 1st generation and 0.32 in the 2nd generation; finally genotype BB was 0.30 in 1st generation and 0.27 in 2nd generation. Frequency of A/B allele in the 1st generation was (0.5A/0.5B); the frequency of the A/B allele in the 2nd generation was 0.53A/0.47B. Figure 3.3. Genotyping at PIT1polymorphic site by RasI M: Standard DNA scale 100 bp The GH gene polymorphism was identified using the restriction enzyme FokI. The results of electrophoresis analysis showed that in the studied pig population, there were three genotypes: homozygous AA genotype corresponding to a 605 bp electrophoresis band, homozygous GG genotype corresponding to two electrophoresis bands which were 260 bp and 345 bp and the heterozygous AG genotype for three electrophoresis bands of 605 bp,345 bp and 260 bp, respectively. The results of determining genotype and allele frequencies of GH gene polymorphisms in Duroc pig population showed that in the 1st generation, the AA genotype had the lowest rate of 0.15 while the GG genotype was 0.35 and AG genotype had the highest rate of 0.50. The A and G alleles had frequencies of 0.40 and 0.60, correspondingly. In the 2nd generation, the AG genotype had the highest rate of 0.44, followed by the GG genotype (0.41) and the lowest was the AA genotype (0.15). The A and G alleles had frequencies of 0.37 and 0.63, respectively. 9
13. Figure 3.4. Genotyping at GH polymorphic site by FokI. M: Standard DNA scale 100 bp The sequencing of the GH gene polymorphisms was shown in Figure 3.5. GenotypeAG GenotypeGG GenotypeAA Figure 3.5. Sequencing results of GHgene polymorphisms 10
14. PCR-RFLP method was used to identify LEP gene polymorphisms. The analysis results of the studied population only obtained 2 genotypes. The CT genotype corresponded to 3 electrophoresis bands of 230 bp, 186 bp and 44 bp and the TT genotype corresponded to one 230 bp electrophoresis band. The results of determining the genotype and allele frequencies of the LEP gene polymorphism in the Duroc pig population showed that the T allele was dominant with a frequency of 0.98 in the 1st generation and 0.97 in the 2nd generation. Figure 3.6. Genotyping at LEP gene polymorphisms by HinfI M: Standard DNA scale 100 bp The sequencing of the LEP gene polymorphisms was shown in Figure3.7. GenotypeTC (reverse direction was genotype AG) GenotypeTT (reverse direction was genotype AA) Figure 3.7. Sequencing results of LEP gene polymorphisms When analyzing the C2604T polymorphism on the PIK3C3 gene fragment belonging to exon 24, chromosome 7 by the restriction enzyme Hpy8I, three genotypes were identified including TT genotype corresponding to a 102 bp electrophoresis band, heterozygous CT 11
15. genotype corresponding to 3 electrophoresis bands 102 bp, 67 bp and 35 bp and the homozygous CC genotype correspondsing to two bands of 67 bp and 35 bp electrophoresis. The results of determining the genotype and allele frequencies of the PIK3C3 gene polymorphism in the Duroc pig population showed that the C allele was dominant with a frequency of 0.62. TT CT CC TT CT CC CC CT M 102bp 100bp 67bp 60bp 35bp 40bp Figure 3.8. Genotyping at PIK3C3 gene polymorphisms by Hpy8I M: Standard DNA scale 50 bp The sequencing of the PIK3C3 gene polymorphisms was shown in Figure3.9. GenotypeCT GenotypeTT GenotypeCC Figure 3.9. Sequencing results of PIK3C3 gene polymorphisms 12
16. 3.3. ASSOCIATION OF MC4R, PIT1, GH, LEP AND PIK3C3 GENE POLYNOLOGIES WITH GROWTH PERFORMANCE, BACKFAT THICKNESS 3.3.1. Association of MC4R gene with growth performance and backfat thickness The influence of factors on the growth performance of Duroc pigs was presented in Table 3.2. Table 3.2. The association of MC4R genotype with growth parameters AA AG GG Generation Parameters p n LSM ± SE n LSM±SE n LSM±SE Pre-test weight (kg) 80 31.58 ± 0.44 254 31.71 ± 0.29 166 31.70 ± 0.34 0.96 Post-test weight (kg) 80 98.61a ± 0.11 254 94.86b±0.69 166 93.15b ± 0.80 0.00 Growth performance 1 80 853.26a ± 9.59 254 820.40b ±6.36 166 790.44c ± 7.31 0.00 (g per day) Backfat thickness (mm) 80 12.62 a ± 0.29 254 11.95 a ± 0.19 166 11.38 b ± 0.22 0.00 Pre-test weight (kg) 32 31.23a ± 0.52 91 30.00ab ± 0.31 65 29.45b ± 0.35 0.02 a b b Post-test weight (kg) 32 101.76 ± 1.88 91 94.54 ± 1.15 65 92.47 ± 1.29 0.00 Growth performance 2 32 860.31a ±15.91 91 814.89b ±9.73 65 797.72b ±10.96 0.00 (g per day) Backfat thickness (mm) 32 12.85a ± 0.59 91 11.48a ± 0.36 65 10.04b ± 0.41 0.00 In the same indicator, LSM values have different letters, the difference was statistically significant (p<0.05) Genotype MC4R was associated with growth performance and backfat thickness (p<0.05) in the 1st and 2nd generation. Growth performance in Duroc pigs which had genotypes AA; AG; GG in generation 1 was 853.26, 820.40, 790.44 g/dayrespectively; was highest in pigs carrying genotype AA and lowest in pigs carrying genotype GG. Duroc pigs carrying genotype AA had backfat thickness of 12.62 mm, reaching the highest backfat thickness and the lowest backfat thickness among pigs carrying genotype GG with 11.38 mm. In the 2nd generation, Duroc pigs carrying genotype AA had the highest growth performance (860.31 g/day), backfat thickness (12.85 mm) and the lowest in pigs carrying genotype GG (797.72 g/day; 10.04 mm). In conclusion, MC4R gene polymorphism was strongly associated with growth performance and backfat thickness in both generations (p<0.05). In which, pigs carrying genotype AA achieved the highest growth performance and backfat thickness in both generations. Generation 1 was 853.3 g/day and 12.62 mm, generation 2 was 860.3 g/day and 12.85 mm. 3.3.2. The association of the PIT1 gene with growth performance and backfat thickness The results of analysis of the association between polymorphisms and growth parameters of Duroc pigs were shown in Table 3.3. 13
17. Table 3.3. The association of PIT1 genotype with growth parameters AA AB BB Generation Parameters p n LSM ± SE n LSM ± SE n LSM ± SE Pre-test weight (kg) 14 31.23b±0.36 202 32.36a±0.29 149 32.15ab±0.37 0.02 a a b Post-test weight (kg) 14 96.27 ± 0.86 202 95.60 ±0.69 149 93.36 ±0.88 0.02 1 Growth performance a ab b (g per day) 14 833.10 ±8.00 202 816.41 ±6.41 149 807.89 ±8.20 0.04 Backfat thickness (mm) 14 12.42a±0.24 202 11.81 ab±0.19 149 11.58b±0.24 0.01 Pre-test weight (kg) 61 30.23±0.36 78 29.89±0.34 49 29.95±0.42 0.75 2 Post-test weight (kg) 61 98.29a±1.22 78 95.55a±1.17 49 89.50b±1.45 0.00 Growth performance a b b (g per day) 61 844.70 ±10.25 78 811.62 ±9.82 49 782.93 ±12.16 0.00 Backfat thickness (mm) 61 12.37a± 0.40 78 11.43a±0.38 49 9.62b±0.47 0.00 In the same indicator, LSM values have different letters, the difference was statistically significant (p<0.05) GenotypePIT1 was associated with growth performance and backfat thickness in the 1st and 2nd generation (p<0.05). Specifically, the traits of growth performance and backfat thickness in the 1st generation was highest in pigs carrying genotype AA (833.10 g/day; 12.42 mm), then genotype AB (816.41g/day; 11.81 mm) and finally genotype BB (807.89 g/day; 11.58 mm). In the 2nd generation, genotype AA still achieved the highest growth performance and backfat thickness, the lowest was for pigs carrying genotype BB at 844.70 g/day compared with 782.93 g/day; it was +61.77 g/day higher in growth performance; 12.37 mm compared to 9.62 mm, was +2.75 mm higher in backfat thickness. Growth performance and backfat thickness were different between the two genotypes AA and BB in Duroc pig population (p<0.05). In conclusion, PIT1 gene polymorphism was strongly associated with growth performance and backfat thickness in both generations (p<0.05). Growth performance and backfat thickness were highest in pigs carrying genotype AA in both generations: Generation 1 was 833.1 g/day; 12.42 mm, the second generation was 844.70 g/day; 12.37 mm. 3.3.3. The association of the GH gene with growth performance and backfat thickness Growth performance of Duroc pigs according to the GH genotype was presented in Table 3.4. Table 3.4. The association of GH genotype with growth parameters AA AG GG Generation Parameters p n LSM±SE n LSM±SE n LSM±SE Pre-test weight (kg) 75 32.05±0.47 252 31.75±0.29 173 31.89±0.33 0.82 Post-test weight (kg) 75 95.73ab±1.10 252 94.23b±0.69 173 96.52a±0.79 0.03 1 Growth performance ab b a (g per day) 75 818.34 ±10.13 252 809.00 ±6.37 173 832.33 ±7.27 0.01 Backfat thickness (mm) 75 12.57a±0.30 252 12.02ab±0.19 173 11.48b±0.22 0.00 Pre-test weight (kg) 78 30.37±0.34 82 29.49±0.34 30 30.40±0.51 0.10 Post-test weight (kg) 78 92.31b±1.19 82 95.24b±1.18 30 101.90a±1.78 0.00 2 Growth performance a b a (g per day) 78 835.74 ±10.15 82 788.50 ±10.10 30 839.93 ±15.16 0.00 Backfat thickness (mm) 78 12.09a±0.39 82 11.34ab±0.39 30 9.97b±0.59 0.01 In the same indicator, LSM values have different letters, the difference was statistically significant (p<0.05) 14
18. The GH gene polymorphism was associated with growth performance and backfat thickness in the Duroc pig population in both studied generations (p<0.05). Pigs carrying genotype GG had the highest growth performance in the 1st and 2nd generation, respectively, at 832.33 g/day; 839.93 g/day, the lowest was pigs carrying genotype AG 809.00 g/day; 788.50 g/day. Growth performance was different between 2 genotypes GG and AG in Duroc pig population (p<0.05). The GH gene polymorphism was closely associated with backfat thickness (p<0.05) in both generations. Backfat thickness was the highest in pigs carrying genotype AA, the lowest in genotype GG in both generations, such as: pigs carrying genotype AA (12.57 mm); AG (12.02 mm); GG (11.48 mm) in the 1st generation. In the 2nd generation, backfat thickness of the genotypesAA; AG; GG were 12.09 mm; 11.34 mm; 9.97 mm. Backfat thickness was different between AA and GG genotypes in Duroc pig population (p<0.05). The GH gene polymorphism was strongly associated with growth performance and backfat thickness in both generations (p<0.05). Pigs carrying genotype GG had the highest growth performance in the 1st and 2nd generation, at 832.33 g/day; 839.93 g/day in that order. The highest backfat thickness in pigs carrying genotype AA in generation 1 and 2 was 12.57 mm; 12.09 mm, respectively. 3.3.4. The association of LEP gene with growth performance and backfat thickness The results of analysis of the association between polymorphisms and growth parameters of Duroc pigs were shown in Table 3.5. Table 3.5. The association ofLEP genotype with growth parameters TT CT Generation Parameters p n LSM±SE n LSM±SE Pre-test weight (kg) 479 32.40±0.23 21 34.07±0.88 0.06 Post-test weight (kg) 479 95.09b±0.57 21 100.59a±2.20 0.01 1 Growth performance 479 817.13b±5.31 21 870.65a±20.24 0.01 (g per day) Backfat thickness (mm) 479 12.92±0.61 21 11.89±0.16 0.09 Pre-test weight (kg) 177 29.93±0.26 11 30.78±0.78 0.31 b a Post-test weight (kg) 177 94.05 ±0.95 11 102.72 ±2.87 0.00 Growth performance 2 177 807.44b±7.75 11 884.23a±23.42 0.00 (g per day) Backfat thickness (mm) 177 11.31±0.31 11 11.40±0.94 0.93 In the same indicator, LSM values have different letters, the difference was statistically significant (p<0.05) Research on the association of LEP gene polymorphism with growth performanceshowed that LEP gene polymorphism was associated with growth performance(p<0.05). The results of analysis of the association of LEP gene polymorphism with growth performanceshowed that growth performanceof pigs carrying genotype CT was higher than pigs carrying genotypeTT, respectively 870.65 g/day compared with 817.13 g/day in the 1st generation; 884.23 g/day compared with 807.44 g/dayin the 2nd generation. In this study,the link between LEP gene polymorphism and backfat thickness was not found. 15