Genetic diversity and productivity of the Cay Cum chicken, special native chicken breed of Vietnam

Thom B T, Phung T V and Hoan N D

Thai Nguyen University of Agriculture and Forestry, Tan Thinh Ward, Thai Nguyen City 50000, Vietnam
ndhoan@tnu.edu.vn

Abstract

A total of 287 blood samples were collected from 6 native chicken populations (Dong Cao, Mia, Ri, Tau Vang, Nhieu Cua, and Cay Cum) to analyze the genetic diversity of Cay Cum (CC) chicken by using microsatellite molecular indicators. To evaluate productivity, a total of 300 one-day-old birds of Cay Cum chicken were randomly allocated to 2 groups with 3 replicates. Birds from the first group are raised by the free-range method in a way that imitates local households (Natural group). The formulated diets were based on locally available ingredients such as corn, grain, rice bran, dry cassava, soybean. Birds from 2 group (Semi-natural group) was raised by semi-free-range method, using the same basic feed as the group 1, however soybean meal was added. Chickens in both groups are raised for up to 20 weeks to monitor the growth performance indicators. After 20 weeks continue raising until 72 weeks of age to monitor reproductive indicators. The CC chicken population has high genetic diversity, average observed heterozygosity (0.61), and expected heterozygosity (0.66), low inbreeding coefficient (0.06). The Cay Cum chicken has a very long genetic distance compared to other indigenous chickens, thereby showing that this chicken is a separate genetic source. Traditional raising methods in households have not fully promoted the potential of Cay Cum chicken. Improving the quality of feed has increased growth performance, egg production, and reduced mortality rate in both broilers and breeders.

Keywords: genetic resources, indigenous chicken, local chicken, microsatellites

Introduction

The importance of native and local breeds of birds for the rural economy in developing and underdeveloped countries mostly in Asia and Africa is very high. They are part of a balanced farming system that has vital roles in the rural households as a source of high-quality animal protein and emergency cash income and play a significant role in the socio-cultural life of the rural community and woman empowerment (Mahendra Kumar Padhi 2016). This is so because they are well-adapted to the extensive husbandry (scavenging) conditions, with very low levels of inputs, under which they are maintained, usually together with other domestic animals. However, the vast majority of these breeds are not or poorly described. In addition, according to The State of the World's Animal Genetic Resources for Food and Agriculture, about 40% of avian breeds are of unknown risk status. Thus, considerable efforts are needed in order to evaluate these breeds. Due to favorable climatic and ecological characteristics, Southeast Asian countries are endowed with a variety of livestock breeds, especially indigenous chicken breeds(Bett et al 2014) and this is also the reason for Vietnam to become one of the largest chicken domestication centers in the world (Ming-Shan Wang et al 2020). In Vietnam, native chicken breeds currently account for 60 - 65% of the total chicken flock. They are of special significance not only in terms of genetic diversity but also bringing economic benefits to farmers in rural and mountainous areas. Therefore, the research to conserve these chicken breeds is very necessary (FAO 2008). Previous studies have shown that in Vietnam there are about 25-30 different native chicken breeds and widely distributed throughout the country, especially in the northern mountainous and central highland provinces. There are 18 chicken breeds recognized and approved by the State: Ri, Mia, Ho, Dong Cao, Tau Vang, Tre, Choi, Te, Ac, H'Mong, Nhieu Cua, Mong, Long Xuoc, Ri’Ninh Hoa, Lac Thuy, H're, Lien Minh, Lac Thuy (Ministry of Agriculture and Rural Development 2018). Cay Cum chicken has been raised for a long time in the Northern provinces (Ha Giang and Cao Bang) of Vietnam. They have a special characteristic that they appear to have no pygostyle. They are chicken kinds with very good meat quality, used in good luck rituals, and a sacred object in the H'Mong family. Cay Cum chicken has been funded by the State for research since 2008, however has not yet assessed the genetic differences of this chicken with other native chickens. To evaluate genetic diversity and kinetic relationships among biological populations, morphological, biochemical, and molecular indicators can be used (Dessie et al 2012), in which the biomolecular indicators are used with many outstanding advantages such as high accuracy, an unlimited number of indicators, independent of the environment and tissue (Pham 2013). So far, there are many types of molecular markers used in genetic diversity research such as RFLP, RAPD, AFLP, minisatellite, microsatellites, mitochondrial DNA sequence analysis (Cuc et al 2010; Dessie et al 2012; Pham 2013). In particular, the use of microsatellites has many advantages such as having many loci in the genome, high length polymorphism, many alleles in one locus, average cost. Therefore, the microsatellite indicator was quickly used in population genetic studies (Cuc et al 2011). The purpose of this study is to evaluate the nature of the genetic structure and production potential of the native CC chicken, thereby contributing to preserving and conserving precious genetic resources, and diversifying chicken breeds in Vietnam.

Materials and methods

Appearance characteristics and current situation of the Cay Cum chicken population raised in the locality

Conducted a survey of 178 households raising CC chicken in Cao Bang and Ha Giang provinces to determine indicators such as appearance characteristics, the flock size, breeding methods, types of feed used, and purposes of CC chicken raising.

Analysis of genetic diversity of Cay Cum chicken by microsatellite molecular index

Table 1. Description of morphological appearance of the native chicken populations used in the study
No	Population of chicken	Photo	Settlement area
1	Cay Cum (CC)		Northern mountainous region (Ha Giang, Cao Bang province)
2	Dong Cao (DC)		Northern delta provinces (Bac Ninh, Hung Yen, Thai Binh)
3	Mia (M)		Northern delta provinces (Hoa Binh, Ha Noi, Thai Binh)
4	Ri (R)		All the country, especially the Northern provinces
5	Tau Vang (TV)		All the country, especially the Southern provinces
6	Nhieu Cua (NC)		Northern mountainous region (Phu Tho, Lang Son provinces)

A total of 287 blood samples were collected from six native chicken populations (Table 1.), including 40 samples from CC, 55 samples of each from DC, M, R, and TV chicken, and 27 samples from NC chicken. Blood samples were collected from the wing vein and stored in EDTA solution at - 20⁰C. Total DNA was extracted from blood samples using the Qiage's DNA extraction kit (Germany). The concentration and purity of the DNA samples after extraction were checked by electrophoresis on 1% agarose gel and on a spectrophotometer. Twenty pairs of microsatellite primers were used to evaluate the genetic characteristics of chicken populations and standardized in five multi-primer PCR reactions; 1µl of all multiplex-PCR products after 25 µl addition of Sample Loading Solution Beckman Coulter buffer and 0.15µl of Beckman Coulter standard size DNA scale isolated by capillary electrophoresis system on automated sequencing CEQ8000 by Beckman Coulter. Allele sizes were analyzed automatically by Genetics analysis system software on CEQ8000 version 9.0. Conducted monitoring the number of alleles, the average number of alleles per locus, average observed heterozygosity (H_O) and expected heterozygosity (H_E), the F-statistic values according to Wright (Wringt' F-statistics), genetic distance, inbreeding coefficient Fis.

Growth performance and reproductive of Cay Cum chicken

A total of 300 one-day-old birds of Cay Cum chicken were randomly allocated to 2 groups with 3 replicates per treatment and 50 chickens in each replicate. Birds from the first group are raised by the free-range method in a way that imitates local households (Natural group). The formulated diets were based on locally available ingredients such as corn, grain, rice bran, dry cassava, soybean (Table 2.). To prevent some common diseases, all the birds are used with vaccines of Smallpox, Gumboro, and Newcastle. Birds from 2 group (Semi-natural group) was raised by semi-free-range method, using the same basic feed as the group 1, however soybean meal was added. Chickens have used 3 vaccines similar to group 1. The chicken barn is made from local materials, with a parking space for chickens to sleep at night and on rainy days. Chickens in both groups are raised for up to 20 weeks to monitor the following indicators survival rate for 0-20 weeks period, body weight, slaughter criteria, and feed consumption. After 20 weeks of age, the low-quality hens are discarded, chicken kept for up to 72 weeks of age to monitor reproductive indicators such as the age of first egg laying, laying rate, egg yield, egg weight, and incubation criteria.

Statistical analysis

The number of alleles, the average number of alleles per locus, average observed heterozygosity (HO) and expected heterozygosity (HE), the F-statistic values according to Wright (Wringt' F-statistics). Genetic distance, inbreeding coefficient Fis were estimated by Genetix software. Analysis of genetic relationships, the pairwise Fst distances genetic between chicken populations using Population and Treeview software. Genetic balance according to Hardy-Weinberg law of each locus was checked using Genepop software. The polymorphic information (PIC) value of each locus is calculated by:

pi, pj: frequency of alleles; i, j, and k: the number of alleles for each locus

Use SAS 9.4 software and PROC MIXED of Statistical Analysis System (SAS Institute, Cary, NC, USA) to calculate growth performance and reproductive indicators. The Tukey test was used for determining the difference between the mean values between groups. The statistically significant difference was determined when P < 0.05.

Results and discussion

The appearence of CC chicken

The male has a more showy color than the hens, mainly purplish red, black red with yellow; hens with brown feathers and sparrow. The stature of the CC Chicken is average compared to other native Vietnamese breeds, the male is about 1600 - 1700g, the female is about 1440 - 1500g / head. The most difference in the appearance of the CC chicken compared to other native chickens is the tail feathers of both the male and the female are short and down, so on the outside, it has not pygostyle and uropygial gland. However, the surgery showed that this chicken still has a uropygial gland in the tail but is smaller in size than other native breeds. The uropygial gland, informally known as the preen gland or the oil gland, is a bilobed sebaceous gland possessed by the majority of birds. It is located dorsally at the base of the tail (between the fourth caudal vertebra and the) and is greatly variable in both shape and size. Some or all species in at least nine families of birds lack a uropygial gland, mostly the ones unable to fly or the ones that produce powder down for feather maintenance (Montalti and Salibian, 2000). The role of the uropygial gland dynamically varies during the annual cycle, potentially in response to seasonal variation in parasitic infection risk ( Orsolya Vincze et al 2013).

Figure 1. The appearence of Cay Cum chicken

Cay Cum chicken population is being raised locally

Survey results of 178 households raising CC chicken in Cao Bang and Ha Giang provinces show that all households raise small-scale chickens with 2 to 23 birds / household, on average 6.2 heads / household. The majority of households keep CC chickens together with other indigenous breeds, affecting the purity of each breed, although up to 76% of households have exchanged cocks to limit inbreeding. Due to increased market demand, the number of CC chickens has tended to increase in recent years, the number of CC chickens in 2020 has increased by 3.0-3.5 times compared to 2016. Most of the households raise by traditional methods, free-range, do not have a barn, use local food sources such as maize, paddy, and soybean seeds. Chickens are not vaccinated and treated when sick. Due to the lack of application of new technologies in raising and controlling diseases, CC chicken raised in households have a high mortality rate, low growth rate, at 20 weeks of age, the mortality rate is about 15%, the average weight of males about 1650g, and females about 1360g. Egg yield is 35 - 40 eggs/hen if hatching naturally, 55 - 60 eggs/hen if not hatched. There is a difference among households in terms of the purpose of raising CC chicken: 84.27% of households sell broiler chicken, 11.24% sell eggs, only 4.49% sell breeding chicken. Survey results on CC chicken breeds are equivalent to some other indigenous chicken breeds naturally raised in households such as Ri chicken, NC chicken, Mia chicken (Su et al 2009).

Genetic diversity of Cay Cum chicken

The analysis results of 20 microsatellite loci out of a total of 287 samples of 6 native chicken populations obtained 238 alleles, an average alleles number is 11.9 per locus, in which, the total number of alleles obtained from the CC chicken is 124 alleles, the average alleles number per locus is 6.2. This result is higher than previous studies on Taiwan and Vietnamese chicken breeds (Cuc et al 2011; Pham et al 2013). The total number of alleles analyzed in CC chicken was lower than that of other chickens such as M chicken (153), TV chickens (155), and NC chicken (140). Besides, the allele frequency of each locus in CC chicken is also different from other breeds, that is, there are 15 alleles appearing only in CC chickens such as allele 151 in MCW216 locus, allele171 in MCW014 locus, alleles 251 and 259 in locus LEI234, alleles 285, 339, 453 and 461 in locus LEI192. The appearance of the characteristic alleles and the difference in allele frequencies of the microsatellite loci in each breed were factors that caused genetic differences between varieties (Maw et al 2015)

The average number of alleles identified in the populations ranged from 6.2 to 7.7, of which in the CC chicken was 6.2 (Table 3). The average observed heterozygosity (Ho) ranged from 0.54 to 0.61, in which the highest was observed in the CC chicken (0.61). The inbreeding coefficient of the CC chicken was 0.06, lower than that of other varieties. Thereby shows that the genetic diversity of chickens Cay Cum is relatively high. This can be explained by the fact that the CC chicken populations are raised in different geographical regions, so they retain genetic diversity, on the other hand, reduce inbreeding between strains.

The results in Table 4. and Figure 2. show that the genetic distance between CC chicken and other native chicken populations ranged from 0.260 to 0.411, of which the genetic distance of CC chickens was closest to NC chickens (0.260), the farthest from DC chicken (0.411). The genetic distance between the 4 breeds of chickens DC, R, M, and TV is very small from 0.057 to 0.141. Opposite, the genetic distance between CC chicken and the remaining 5 breeds is very far, which proves the genetic differences of CC chicken compared to others. This is a very meaningful result in building a plan for the conservation and development of the CC chicken breed in the future. The results of this study were consistent with the publication of Berthouly-Salazar et al (2010) that in contrast to other chicken populations, the Ha Giang chicken population showed very high genetic diversity at both the nuclear and mitochondrial levels. Cuc and Weigend (2019) showed that within genetic variability of Vietnamese local chicken was high compared to another chicken group except for Vietnamese exotic breeds of Chinese origin. The Vietnamese local populations closely clustered with the Red Jungle fowl population. Due to its past and recent history, this population accumulates a specific and rich gene pool highlighting its interest and the need for conservation.

Figure 2. Neighbor tree of 6 native chicken populations constructed using Nei’s genetic distance

Growth performance and reproductive of CC chicken

CC chicken are raised by traditional methods (without barns, using local ingredients that are poor in nutrition, especially lacking in protein) have high mortality, low growth, and reproduction performance. Improved feed quality (Semi-natural group) has reduced mortality, increased growth performance, and reproduction of CC chicken (Table 5. and Table 6.). The mortality of CC chicken in both groups was mainly at the first week (9% for the Natural group and 6.33% for the Semi-natural group). At 20 weeks of age, the mortality rate of the Natural group was 13%, 4.33% higher than that of the Semi-natural group (8.67%). The body weight (BW) of CC chicken at 1 week of age for the 2 groups was similar. Then, BW in the Semi-natural group was always higher than that in the Natural group (P< 0.05). At 20 weeks of age, BW of the Semi-natural group was 1697.52 g for males and 1439.78 g for females, which was statistically significantly higher (P < 0.05) than of the Natural group (1594.23 and 1360.42 respectively). The carcass ratio of chickens in the Natural group was 65.45 %, which was statistically significantly lower (P < 0.05) than that of chickens in the Semi-natural group (67.79%). Other indicators such as breast meat, thigh meat, and abdominal fat of chickens in the two groups were similar. The FCR of both groups was higher than that of industrial chicken farming. Even so, the birds of the group that received the high-protein feed (Semi-natural group) had a significant decrease in FCR compared to the Natural group (4.64 compared to 5.08).

The mortality rate of CC chicken at the reproductive period was 6.67% to 10.83% which is average compared to other native breeds. The improvement of feed quality has the effect of accelerating the hens' reproductive development, which has been demonstrated by indicators such as age at first egg, age at 5%, age at 50% of laying rate, and age at highest laying rate in the Semi-natural group were all earlier than that in the Natural group. The number of eggs/year/hen in the Semi-natural group was 75.84 eggs, significantly higher than that in the Natural group 46.56 eggs (P < 0.05 ). Other indicators such as egg weight, fertility, and hatchability of chickens in the 2 groups are similar. Similar to FCR for growth, FCR for egg production of CC chickens is also quite high when new technology is not applied. The FCR of the Semi-natural group was 2.53g of feed / g of egg, lower than the Natural group of 3.50 g of feed / g of the egg (P< 0.05). The results of this study are consistent with the publication of Nguyen (2016).

Conclusions

• The Cay Cum chicken population has high genetic diversity, average observed heterozygosity (0.61), and expected heterozygosity (0.66), low inbreeding coefficient (0.06). The Cay Cum chicken has a very long genetic distance compared to other indigenous chickens, thereby showing that this chicken is a separate genetic source, so the conservation and generation of the genetic source of the Cay Cum chicken are very necessary.
  
  
• Traditional raising methods in households have not fully promoted the potential of Cay Cum chicken. Improving the quality of feed has increased growth performance, egg production, and reduced mortality rate in both broilers and breeders.

Conflict of interest

We acknowledge that there is no conflict of interest with any person or entity associated with the manuscript of this article.

Acknowledgement

The authors gratefully acknowledge funding of the Ministry of Science and Technology, Vietnam . We also would like to thanks to Institute of Life Science, the Thai Nguyen University to facilitate the experiment.

References

Berthouly C, G Leroy, V T Nhu, H H Thanh and B Bed'Hom 2009 Genetic analysis of local Vietnamese chickens provides evidence of gene flow from wild to domestic populations. BMC Genetic10: 1. https://doi.org/ 10.6845/NCHU.2013.01123

Berthouly-Salazar C, X Rognon, T Nhu Van, M Gély, C Vu Chi, M Tixier-Boichard, B Bed'Hom, N Bruneau, E Verrier, J C Maillard and J R Michaux 2010 Vietnamese chickens: a gate towards asian genetic diversityBMC Genetics 11: 53. https://doi.org/ 10.1186/1471-2156-11-53.

Besbes B 2009 Genotype evaluation and breeding of poultry for performance under suboptimal village conditions.World's Poultry Science Journal 65: 265-271. https://doi.org/doi:10.1017/S0043933909000221

Besbes B, M Tixier-Boichard, I Hoffmann and G L Jain 2008 Future trends for poultry genetic resources. Proceedings of the International Conference of Poultry in the 21st Century: Avian Influenza and Beyond. Bankok 2007: 5-7. http://www.fao.org

Bett R C, A K F H Bhuiyan, M S Khan, G L L P Silva, Le Thi Thuy, S C Sarker, M N D Abeykoon, Thi T H Nguyen, S Sadef, E Kariuki, I Baltenweck, J Poole, O Mwai and M N M Ibrahim 2014 Indigenous chicken production in the South and South East Asia. Livestock Research for Rural Development 26: 229. http://www.lrrd.org/

Cuc N T K, H Simianer, H Eding, H V Tieu and V C Cuong 2010 Assessing genetic diversity of Vietnamese local chicken breeds using microsatellites. Animal Genetic 41: 545–547. https://doi.org/10.1111/j.1365-2052.2010.02039.x

Cuc N T K, Weigend S, H V Tieu and H Simianer 2011 Conservation priorities and optimum allocation of conservation funds for Vietnamese local chicken breeds. Journal of Animal Breeding Genetic 128: 284–294. https://doi.org/ 10.1111/j.1439-0388.2010.00911.x

Cuc N T K and Weigend S 2019 Genetic Relationship Between Vietnamese Chicken Populations and Chicken Populations from Different Continents. Animal and Veterinary Sciences 7 (4): 94-101. https://doi.org/10.11648/j.avs.20190704.12

Dessie T, N Dana, W Ayalew and O Hanotte 2012 Current state of knowledge on indigenous chicken genetic resources of the tropics: domestication, distribution and documentation of information on the genetic resources. Worlds Poultry Science Journal 68: 11–20. https://doi.org/10.1017/S0043933912000025

FAO 2008 Poultry production systems in Vietnam; GCP/RAS/228/GER. Working Paper No. 4, Pages: #22. http://www.fao.org

Mahendra Kumar Padhi 2016 Importance of Indigenous Breeds of Chicken for Rural Economy and Their Improvements for Higher Production Performance; Scientifica 6: 1-9. https://doi.org/10.1155/2016/2604685

Maw A A, K Kawabe, T Shimogiri, W Rerkamnuaychoke, Y Kawamoto, S Masuda and S Okamoto 2015 Genetic diversity and population structure in native chicken populations from Myanmar, Thailand and Laos by using 102 indels markers. Asian-Australia Journal of Animal Science 28(1): 14-9. http://doi.org/10.5713/ajas.14.0212.

Ministry of Agriculture and Rural Development – MARD, Vietnam 2018 Circular No. 01/2018 / TT-BNN & PTNT.

Ming-Shan Wang, Mukesh Thakur and 2020 863 genomes reveal the origin and domestication of chicken. Cell Research 30: 693–701. https://doi.org/10.1038/s41422-020-0349-y

Montalti Diego and Salibian Alfredo 2000 Uropygial gland size and avian habitat. Ornitologia Neotropical. 11 (4): 297–306.

Nei Masatoshi 1972 Genetic distance between populations. The American Naturalist 106(949): 283–292.http://doi.org/10.1086/282771.

Nguyen T D 2016 Study on appearance and productivity of Cay Cum chicken raised in Thai Nguyen. Master thesis , Thai Nguyen University of Agriculture and Forestry, Vietnam.

Orsolya Vincze, Csongor I Vágási, István Kovács, Ismael Galván and Péter L Pap 2013 Sources of variation in uropygial gland size in European birds. Biological Journal of the Linnean Society, 110 (3): 543-563. https://doi.org/10.1111/bij.12139

Pham M H 2013 Analysis of genetic diversity and conservation priorities in Asian domestic chicken population base on microsatellites; Ph.D.Thesis, National Chung Hsing University, Taiwan

SAS 2009 SAS User’s Guide, Statistics. SAS Institute, Inc., Cary, NC. USA

Su V V, Tieu H V and Thieu N C 2009 National Genetic resources of Vietnam indigenous poultry breeds. Journal of Animal Science and Technology 6: 88-94.http://vcn.mard.gov.vn

Tixier-Boichard M, F Leenstra, D K Flock, P M Hocking and S Weigend 2012 A century of poultry genetics. Worlds Poultry Science Journal 68: 307–321. https://doi.org/10.1017/S0043933912000360