A study of reproductive characteristics and gender selection of mong cai pigs in quang ninh

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY THESIS TOPIC: A study of reproductive characteristics and gender selection of Mong Cai pigs in Quang Ninh HANOI, 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY THESIS TOPIC: A study of reproductive characteristics and gender selection of Mong Cai pigs in Quang Ninh Full name : VU HAI YEN Class : K62CNSHE Student code : 620560 Supervisors : Msc NGO THANH TRUNG : Msc NGUYEN QUOC TRUNG HANOI, 2022 COMMITMENT I commit this is the work of myself The data and results mentioned in thesis are honest and have never been published in any other study I commit that the information cited in the thesis have been specified origin Student Vu Hai Yen i ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor, Msc Ngo Thanh Trung and Msc Nguyen Quoc Trung not only for his teachings, guidance, and support but also for the amazing opportunities he afforded me that went beyond the realm of my thesis I also would like to send my appreciation to the other members of my laboratory, who studying in K62CNSHC, K66TYA class have supported and helped me with my research, but I think the most important to me was their friendship Last, I would like to thank my family whose love, unwavering support and encouragement is what has got me this far ii TABLE OF CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ABSTRACT viii CHAPTER INTRODUCTION CHAPTER LITERATURE REVIEW 2.1 Pork demand of the Vietnam market 2.2 Introduction of Mong Cai pig breed 2.3 Demand for seed sources suitable for concentrated livestock planning areas of the province 2.4 Significance of applying sex control technology to increase female in Mong Cai pig herd CHAPTER MATERIALS AND METHODS 23 3.1 Materials 23 3.2 Research duration and location 24 3.3 Experimental designs 25 3.4 Methods 28 3.4.1 Methods of boar native semen quality assessment 28 3.4.2 Method of preparing percoll solution column (A.Valccircel, 1996) 30 3.4.3 Method of preparing BTS extender and bovine serum albumin (BSA) solution column (Johnson, 1988): 33 CHAPTER RESULTS AND DISCUSSION 35 4.1 Selection of experiment Mong Cai boars and sows based on phenotype and reproductive characteristics 35 4.2 Experiment - Assessment of biological characteristics of collected semen after treated by sperm gender separation solution columns 60 iii 4.3 Assesment of artificial insemination efficiency and gender ratio of offsprings using separated sperms 65 CHAPTER CONCLUSIONS AND RECOMMENDATIONS 68 5.1 Conclusions 68 5.2 Recommendations 69 REFERENCES 70 iv LIST OF TABLES Table 4.1 List of 20 potential Mong Cai boars with breed-specific phenotypes 36 Table 4.2 List of 80 potential Mong Cai saws with breed-specific phenotypes 43 Table 4.3 Semen quality parameters of experiment boars 56 Table 4.4 Semen quality parameters of collected semen layer after separated through percoll concentration gradient column 62 Table 4.5 Semen quality parameters of collected semen layer after separated through bovine serum albumin concentration gradient column 64 Table 4.6 Litter size and gender ratio of piglet generations by artificial insemination using collected semen doses 66 Table 4.7 Litter size and gender ratio of piglet generations by artificial insemination using collected semen doses 66 v LIST OF FIGURES Figure 3.1 Appearance of Mong Cai sows 23 Figure 3.2 Appearance Mong Cai boars and semen collection 23 vi LIST OF ABBREVIATIONS A Sperm motility C Sperm concentration (million sperm / ml) V Semen volume (ml) K Sperm abnormality rate (%) Minute AI Artifical Insemination BTS Beltsville Thawing Solution BSA Bovine serume albumin vii ABSTRACT An application study of artificial insemination efficiency and offspring sex ratio using separated sperms through percoll concentration gradient column and bovine serum albumin concentration gradient column in Mong Cai pig The requirements of study are (i) Selection of experiment Mong Cai boars and sows based on phenotype and reproductive characteristics, (ii) Evaluating sperm concentration and motility of collected sperm layer after separated through discontinuous bovine serum albumin concentration gradient column and percoll concentration gradient column and (iii) Evaluating litter size and gender ratio of piglet generations carried out by artificial insemination using collected semen after separated through discontinuous bovine serum albumin concentration gradient column and percoll concentration gradient column experiments are designed: (i) experiment - Investigation and selection of Mong Cai pigs with purebred phenotype and assessment of Mong Cai boar native semen quality, (ii) Experiment - Assessment of biological characteristics of collected semen after treated by sperm gender separation solution columns (iia) - Experiment 2a Assessment of biological characteristics of collected semen after treated by discontinuous percoll concentration gradient column, (iib) - Experiment 2b Assessment of biological characteristics of collected semen after treated by discontinuous bovine serum albumin (BSA) concentration gradient column; (iii) Experiment Assesment of artificial insemination efficiency and gender ratio of offsprings using separated sperms, (iiia) - Experiment 3a Assesment of artificial insemination efficiency and gender ratio of offsprings using separated sperms through discontinuous percoll concentration gradient column, (iiib) - Experiment 3b Assesment of artificial insemination efficiency and gender ratio of offsprings using separated sperms through discontinuous bovine serum albumin (BSA) concentration gradient column The results are (i) 20 individuals were selected, aged between 12 and 24 months old out of 70 males, whose physical characteristics were average according to the breed standard set for Mong Cai pigs according to Vietnamese standards 9713: 2019 (TCVN 9713:2019), specifically as follows: Balanced body, agile; the middle of viii Sperm concentration (C) is the numbers of sperms in a unit volume of semen Sperm concentration is an important parameter of semen quality assessment, associating with semen volume (V) and sperm activity (A) parameters to calculate the total progressive sperm numbers each ejaculation based on which to decide semen extending ratio and to calculate semen dose numbers for storage or artificial insemination proceeding (Johnson 2000) The average of sperm concentrations of 60 observations of 10 Mong Cai selected boars multiplying with times of replication per boar is 288,93 ± 38,69 millions of sperms / ml According to the other authors, this value is 403 millions of sperms / ml in Duroc breed and 312 million sperm/ml in Yorkshire breed (Spinaci, 2016); 254 million sperm/ml in Duroc x Pietrain breed, 248 in Pietrain x Duroc breed (Spinaci, 2016); 258 in Duroc x Pietrain breed, 251 million sperm/ml in Polish Large White breed and 245 million sperm/ml in Polish Landrace breed (Chaudhary, 2014); 444 million sperm/ml in Duroc breed and 548 million sperm/ml in Pietrain breed (Kondracki 2012) However, compared with the results published in 2013 by Ha Xuan Bo and Than Van Hien, the sperm concentration of Piétrain and Landrace breeds raised in Vietnam was about 175,55 million sperm/ml lower than that of pigs Mong Cai males in this study Compared with the standard TCVN 9713:2019 for Mong Cai pig (sperm concentration > 200 millions of sperms / ml), the quality of sperm concentration of the studied pig breeds satisfied feature Sperm motility is the percentage of progressive sperms of each semen sample observed on microscope or microscope connected with camera associated with computer software (CASA - Computer Associated Semen Analysis) Sperm motility unit in microscope observation and evaluation is point (sperm motility point) ranging from to while in CASA is % of sperm motility on microscope observation field assisted and evaluated by computer The conversion between microscope sperm motility values and CASA is that: point = %, 0,1 = - 15 %, 0,2 = 15 – 25 %, 0,3 = 25 – 35 %, 0,4 = 35 – 45 %, 0,5 = 45 – 55%, 0,6 = 55 – 65 %, 0,7 = 65 – 75 %, 0,8 = 75 – 85 %, 0,9 = 85 – 95 %, = 100% Requirement of entering sperm motility parameter for using boar semen samples to extend and storage in liquid state is > 70 % and sperm cryopreservation is > 80 % (Johnson, 2000) and the accepted values of after 58 incubated or post thawed sperm motility for artificial insemination with liquid storage semen samples are above 60% and above 30 % with cryopreservation semen In this study, sperm motility of 10 selected Mong Cai boars is 88,52 ± 4,47 % Compared with the results of Kondracki et.al in 2012 showed that Mong Cai boars in this study achieved sperm concentration parameter was very good Reasons for this are that Mong Cai pig is a local pig breed which is adapt to the specific climate and environment in Vietnam and the nutrient diet and feeding conditions for experiment dogs are good As reported by authors, sperm motility parameter of boar semen also ranges by seasons Mean of sperm motility values in this experiment is similar to that of also genuine super lean meat pig breeds, white hair pig breeds and hybrid breeds between super lean meat pig breeds and white hair pig breeds in same season: 82 % (0,8) in Duroc breed and 83 % (0,8) in Yorkshire breed (Baker, 1964); 80 % (0,8) in Duroc breed and 0,79 % (0,8) in Pietrain breed (Kondracki 2012) Compared with the standard TCVN 9713:2019 for Mong Cai pig (sperm motility > 70%), the quality of sperm motility of the studied pig breeds satisfied feature Abnormal morphology (%) Rate of abnormal sperm is percentage of abnormal morphology sperms on microscopy observed semen field Abnormal morphology of sperms are the sperms with morphological abnormalities in the head, neck or tail They are not capable of fertilization This is an important parameter of quality assessment at the moment of sperm quality test (Johnson, 2000) If abnormal morphology sperm rate is too high, the fertility rate will be low, the semen sample would be rejected A complete morphological examination is recommended when boars are introduced into the AI centre and during subsequent regular routine examinations In addition to the incidence of cytoplasmic droplets, the percentage of other morphological alterations should not exceed 20% The use of a phase-contrast microscope and glutaraldehyde BTS or formol - citrate fixed wet preparations is recommended, since they avoid the artifacts associated with dried smears (Johnson, 2000) According to requirements of abnormal morphological parameter of boar semen sample, the observed values in this experiment satisfy the requirements: average of this parameter of 120 observations, 60 selected observations, 60 eliminated observations respectively is 13,75 ± 2,45 %; 12,6 59 ± 1,35 %; 14,9 ± 2,81 % These results are higher than as reported by Kondracki 2012 with 1,39 % in Pietrain breed and 1,06 % in Duroc breed because of winter is the test period of this experiment Compared with the results of Hu (2015), these results in this study are slightly higher but still within the permitted range of technical requirements for sperm quality of pig for artificial insemination.compared with the standard TCVN 9713:2019 for Mong Cai pig (abnormal morphology < 15%), the quality of abnormal morphology of the studied pig breeds satisfied feature Sperm resistance (R) is an indicator to assess the resistance of sperm in adverse environmental conditions The scientific basis for assessing the resistance of sperm is the resistance of the lipoprotein membrane of the sperm under the influence of hypertonic NaCl solution The higher the sperm resistance R value, the better the semen quality The average resistance of spermatozoa boars is 3210,37 ± 212,28 for 20 Mong Cai boars; 3292,57 ± 173,99 for 10 selected Mong Cai boars; 3128,16 ± 223,26 for eliminated Mong Cai boar Comparing the results obtained in the Duroc pig breed 4004.2 ± 38.99 with the study of Phan Van Phung, Dang Vu Binh (2008), it was found to be higher, but in the Piétrain breed 7374.10 ± 1682, 27 According to research by Ha Xuan Bo, Do Duc Luc and Dang Vu Binh, the results on resistance of the pig breeds we studied were lower However, compared with the standard TCVN 2839-79 (resistance of foreign pig sperm > 3000), the quality of the pure semen of the studied pig breeds satisfied feature 4.2 Experiment - Assessment of biological characteristics of collected semen after treated by sperm gender separation solution columns Experiment 2a Assessment of biological characteristics of collected semen after treated by discontinuous percoll concentration gradient column According to the results presented in table 4.4, sperm activity of collected semen layers after separated through column (2-4-4 ml; 45%-90%) using centrifuge spermatozoa through a percoll gradient column at 25°C, 1000 rpm, 10 minutes After 10 minutes, layers of sperm were obtained, volume of each layer is 4ml To separate sperms in order to obtain layers containing high percentage of sperm bearing desired sex chromosome to serve manufacturing, firstly the sperms needed to be ensure having 60 certain quantity and good quality to reach the requirements to success of fertilization process There are some parameters for evaluating sperm quality as the integrity of the plasma membrane, sperm agglutination level, total of progressive sperms, sperm concentration, sperm morphology In this experiment, evaluated three parameters are sperm concentration, sperm motility and abnormal morphology In which, from sperm concentration of obtained pig sperms after separated through two types of percoll concentration gradient medium columns we can comparing the total number of separated sperms to initial sperm numbers then calculate the productivity of separating process and predict the sex sperm separating efficiency Of course, to know the exact F-body bearing sperm ratio or X/Y chromosome bearing sperm ratio in any sperm population we have to perform fluorescent staining method The average sperm motility of the sperm population collected from the lower layer is 91,56 ± 3,11%; 70,12 ± 2,16% for upper layer The mechanism involved in the separation of X and Y chromosome bearing spermatozoa using this method rely on the difference in spermatozoa head dimensions and motility It seems that the bottom (X) fraction spermatozoa derived from the 8-layer discontinuous Percoll gradient for sex preselection has higher motility, progression and hyperactivation when compared with the top (Y) fraction spermatozoa The bottom (X) fraction spermatozoa have greater longevity, motility and shorter tails According to the other authors, sperm motility of the sperm population collected from the lower layer is 92,1 ± 2,0% (Wang, 1994), equivalent with result of this study and according to research by Matás (2011), the results on sperm motility of the pig breeds we studied were lower (89,5 ± 5,0%) Then loaded sperm concentration (millions of sperms/ml) is 4100,12 ± 3,2, the results of sperm concentration of collected semen upper layer and lower layer respectively 3600,29 ± 10,54; 380,66 ± 2,5 millions of sperms/ml These results are also suitable with the observed results in study of Matás, 2011 in boar semen In the experiment 2a, the collected sperms from the lower layer after separation (layer 2) of the percoll concentration gradient medium column were in ml in volume Means that from ml native semen we obtain ml in volume of separated sperm 61 containing medium With the obtained sperm concentrations as shown in table 4.4 in the percoll concentration gradient medium column, after separation we have ml x 880,66 millions of sperms = 3522,64 millions of separated sperms in total Means that we have to perform columns using ml of native semen to produce 1,5 dose of separated sperms to use for AI once (2 billions / dose – following the report of Manafi, 2011) Table 4.4 Semen quality parameters of collected semen layer after separated through percoll concentration gradient column Obtained sperm layers Parameters Volume of collected semen layer (ml) Layer Layer (Upper layer) (Lower layer) 4 70,12 ± 2,16a 91,56 ± 3,11b 4100,12 ± 3,2 4100,12 ± 3,2 3150,29 ± 10,54a 880,66 ± 2,5 b 6,13 ± 0,43a 4,05 ± 0,12b Sperm activity of collected semen layers after treatment (%) Loaded sperm concentration (millions of sperms/ml) Sperm collected concentration semen of layer (millions of sperms/ml) Abnormal morphology (%) Note: Difference of upper letters indicate significant difference (P