Department of Agriculture

Philippine Carabao Center

Science City of Muoz, Nueva Ecija

Research Code: BG16001-ROG

Title: Utilization of DNA marker Selection in Breeder and Commercial Farm
Researchers: Sherwin D. Matias, Jonathan C. Pablo and Ester B. Flores
Implementing Agency: Philippine Carabao Center
Funding Agency: Philippine Council for Agriculture, Aquatic and Natural Resources
Research and Development
Budget: P 4,998,389.00
Date Started: October 2016
Expected Date of Completion: September 2018

Introduction

The swine industry is the second largest contributor to the Philippines agricultural sector
coming in next to rice. Swine population inventory in 2015 stood at 12 million head. In
the same year the swine industry contributes about P206 billion to GVA in agriculture,
which was derived from the total volume of production estimated at 2,120.33 thousand
metric tons of hog liveweight. The industry contributes 12 percent of the countrys total
value of agricultural production.
In addition to its contribution to agricultural production and food security, the swine
industry also supports allied industries that provide employment and business
opportunities to millions of Filipinos.
Among the local animal industries, the swine industry is considered as the most organized
and the highest adopter of updated technologies. The application of technologies in
feeding, housing and management and disease prevention and control resulted to
significant improvement in swine growth and production performance. Yet, overall
production efficiency still lags behind major swine producing countries due to low
reproductive performance. Genetic improvement is slow through conventional methods
due to the fact that reproductive traits are lowly heritable and are expressed late in life.
The development of molecular methods of identifying genetic markers provided
opportunity for breeders to hasten the rate of genetic improvement in conjunction with
conventional method of culling and selection. The use of genetic markers associated with
economically important traits had been tried in other countries and has resulted in positive
results to their industry.
Under this assumption an R&D program was successfully implemented by PCC in 2012
in partnership with the Bureau of Animal Industry (BAI) and with the Accredited Swine
Breeders Association of the Philippines (ASBAP). The program has developed and
initially promoted application of genetic markers in the breeding and selection of pigs in
local breeder swine farms. Significant accomplishment of the program was the

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establishment of 17 gene marker protocols for genetic testing of swine and the
establishment of the swine genetic analytical service laboratory (SGASL). Preliminary
estimate of the effect of favorable genotypes of genes related to reproduction through an
association analysis of marker with phenotype indicates an additional of 0.5 1 piglet per
sow per farrowing and the reduction in farrowing interval equivalent to a 3.5% increase
in farrowing index among sows carrying the favorable allele of the leukocyte inhibitory
factor (LIF) gene. With such positive result, the swine industry will benefit from wider
testing of breeder animals as additional information in mating/selection and policy
decisions. The use of the available technology (molecular selection) at the SGASL should
be promoted to nucleus and commercial herds to maximize the benefit that can be derived
from the use of the technology. Thus, a follow through project is being proposed to widen
the application and promotion of the gene marker thru the operationalization of the
SGASL and to hasten the genetic improvement in local breeder and commercial farm
units. Activities that will be pursued under the proposed R&D project would involve
promote the adoption and utilization of the genetic testing technology at wider scale by
the swine raisers. This includes an expanded association analysis to include more traits
and genes in the study. To ensure access of genetic evaluation services by swine breeder
and commercial farms, a commercial genomic laboratory was already established and
currently being operated and managed by the Accredited Swine Breeder Farm
Association of the Philippines (ASBAP), Inc. in coordination with the PCC and BAI. The
collaborating government agencies will implement activities that will promote
sustainability of the project. Among these activities are : R&D for the generation of new
knowledge and technologies on swine molecular genetics, formulation of enabling
policies for swine industry development, monitoring and evaluation of project activities
and accomplishments and other coordination functions that would ensure smooth
implementation of the project and attainment of desired results. In addition to operation
and management of the service laboratory, the ASBAP, Inc. is now promoting the use and
application of the gene marker technologies to active primary players of the local swine
industry.
The expected outputs of the proposed R&D project include wider utilization and adoption
of the gene marker technology by swine raisers, both by breeder and commercial farms.
This higher adoption rate would increase the frequency of favorable genes present in the
local swine population particularly in the breeder pigs.
The 2-year implementation of the proposed R&D project would require a total budget of
Four million nine hundred and ninety nine thousand pesos (P 4.99 M).

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Materials and Methods

Project implementation framework

Implementation of the proposed R&D program is based on the general principles and
protocols of genotyping, analysis of data for marker and phenotype association for different
traits and utilization marker information in animal breeding and selection (Fig. 1).

Figure 1. Sequence of activities involved in the application of molecular genetics

in swine breeding

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Schematic illustration of R&D implementation
The proposed R&D project will be implemented in partnership with the Accredited Swine
Breeders Association of the Philippines (ASBAP), the Bureau of Animal Industry (BAI),
which is acting as the secretariat of ASBAP, the Philippine Carabao Center (PCC), an
agency mandated to lead animal biotechnology R&D, with PCAARRD providing R&D
funds and program monitoring and evaluation services. The proposed project considers the
academe as source of new and novel basic information that would be useful in innovating
current techniques and protocols in animal genetic analysis, interpretation and utilization.
Figure 2 illustrates the implementation scheme and flow of services and R&D outputs.

Figure 2. Schematic illustration of activities and flow of services and R&D outputs
of the proposed R&D program

To ensure smooth implementation of the proposed R&D program specific roles of the
various collaborating agencies and entities are defined. Table 2 presents the expected roles
of each of the collaborating agencies and/or groups.

Table 2. Role of collaborating agencies and/or groups

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 Collaborating
Role
Agencies/Groups
PCAARRD Provide R&D funds
M&E to ensure timely delivery of outputs
Planning and coordination of project activities
Initiate and provide support for collaboration with
foreign laboratories
Packaging and dissemination of information
PCC Lead the implementation of R&D activities e.g. data
analysis/association study and protocols for genetic
evaluation
Provide technical assistance in the operation of the
swine genetic analytical service laboratory
Serve as reference laboratory for genetic analysis
BAI Coordinate related activities of the ASBAP
Lead the formulation of enabling policies
Support the advocacy of the program to swine breeder
farms
ASBAP Provide blood (DNA) samples for R&D purposes
Coordinate with PCC in the implementation of R&D
activities
Lead the operation of the swine genetic analytical
service laboratory
Lead the advocacy on the adoption of genomic
technology to the local swine industry
Academe (i.e. Provide technical support during R&D implementation
UPLB/CLSU) and operation of the genomic service laboratory
Serve as source of basic information needed in the
continuous improvement genetic evaluation and
interpretation protocols.

Animals
Great grand parental (GGP) and grand parental (GP) breeder pigs of the Landrace, Large
White and Duroc breeds and their crosses with production and reproduction records that
are owned by participating members of the Accredited Swine Breeder Association of the
Philippines (ASBAP) will be used as source of genetic materials (DNA) that will be used
by the project. Blood and other appropriate tissue samples will be collected and processed
following protocols adopted by PCC as regards genetic material sample collection,
processing and handling. Collection of blood and other appropriate tissue samples will be
accomplished in accordance with policies and rules pertaining to animal care and welfare.

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Selection of genes to be tested
Selection of genes to be tested per sample submitted in the laboratory will be the
determined by the swine raiser but us limited to the list of test being offered by the
laboratory. These tests are based on the protocols developed by PCC in the Phase I of the
program. Focus of the study will be on the genes that are associated with economically
important positive phenotypes: ESR, PRLR, and LIF for litter size, HFABP, LEPR, and
IGF2 for meat quality, and FUT1 for disease resistance specifically resistance to E. coli
(ETEC F-18).

Sample size
Due to the expected wide allelic variations of genes in a given population, at least 1500
samples will be used in analyzing each of the target genes. DNA samples will be extracted
from blood or other appropriate tissue samples taken from great grand parental (GGP) and
grand parental (GP) breeder pigs owned by participating ASBAP member farms. To
facilitate phenotypic association of genetic characteristics, only pigs that have performance
records will be considered for testing.

Association analysis
Association analysis between phenotype (i.e., litter size total and alive, number of
functional teats, sow index, growth rate, back fat, marbling, etc.) and genotype information
will be conducted to determine the value of the established DNA marker for desired traits.
Analysis of data will be accomplished using appropriate commercially available software.
Allele and genotype frequencies will also be determined for each gene marker used in the
sampled population.
For the the association between a marker and a trait can be tested with single marker
regression as : y = Wb + Xg + e. Where y is a vector of phenotypes, W is a design matrix
assigning phenotype records to fixed effects, b is a vector of fixed effects (e.g. the mean,
population structure. effects, age and so on), X is a design matrix allocating records to the
marker effect, g is the effect of the marker and e is a vector of random deviates eij ~ N(0,
2e), where 2e s is the error variance. In this model the effect of the marker is treated as a
fixed effect, and the model is additive, such that two copies of the second allele has twice
as much effect as one copy, and no copies has zero effect. Software/program to be used for
statistical analysis will be JMP12 (SAS Institute) or R V3.3.1 (http://www.Rproject.org).

Reporting of results and recommendations

Results of tests on individual animals shall only be given to the authorized person identified
by the swine raiser as stated in the request form when samples are submitted to the
laboratory. This is to ensure confidentiality of results. A meeting can be arranged between
the farm and the laboratory staff to discuss the results of the tests and any appropriate
recommendation upon request, as performance records will be requested from farms
submitting samples for testing especially records pertaining to breeding/reproduction,

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growth and carcass quality. Appropriate statistical analysis exclusive for each farm shall
be done and will become part of the report to be discussed. This will include not only the
benefit of the use of markers of positive traits in the breeding program but the limitations
and identifying with the farm owner/managers the various management factors/practices
that may have substantial influence that might interfere with the expected positive results
on the use of markers.

Sustainability activities
To ensure sustainability of using molecular markers in swine breeding and selection the
swine genetic analytical service laboratory will be established and operated commercially
by the ASBAP. The said laboratory will be made available to swine breeding farms for
their genetic analysis needs at reasonable costs. Although cost of genetic evaluation is
estimated at the PCC laboratory to be between P280-350 per sample, toll charges at the
proposed genomic will be adjusted to ensure economic viability of the operation.

Status or progress of the research to date

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As of June 2017 the Second Phase of the study has collected a total of 520 samples from 4
farms that will be used for DNA extraction then testing for the positive genes. Out of these
samples, 280 have been genotyped and analyzed (association analysis between marker and
phenotype) and the remaining samples were already genotyped but still waiting to be
analyzed due pending animal performance records from the farms.
The table below shows the total partial analysis of marker effect for litter size. While this
is just a partial analysis, on the average, sows carrying the favorable allele (B)for the ESR
and LIF gene have a slightly more piglets born live than sows without the favorable allele
(AA).

Table 3. Summary of results per genotype from genes ESR and LIF.

AA AB BB
Parity No. Overall Average N
Mean N Mean N Mean N
ESR
1 10.6 9 12.4 19 11.7 15 11.7 43
2 9.7 9 10.9 19 11.2 15 10.6 43
3 12.6 9 12.2 14 13.5 10 12.7 33
4 12.2 5 11.4 8 10.2 6 11.2 19
5 12.3 3 10.7 7 9.8 5 10.7 15
6 13.5 2 9.3 3 13.8 5 12.4 10
Average/genotype 11.4 37 11.5 70 11.7 56 11.5 163

LIF
1 13 1 11.8 32 11.3 12 11.7 45
2 6 1 11.0 32 10.0 12 10.6 45
3 13.3 24 11.1 9 12.7 33
4 10.9 15 12.3 4 11.2 19
5 10.3 14 17.0 1 10.7 15
6 12.4 10 12.4 10
Average/genotype 9.5 2 11.7 127 11.1 38 11.5 167

Individual Farm Results

Swine Genetic Analytical Service Laboratory (SGASL) conducted a series of tests in Farm
1 in relation to genes affecting litter size and disease resistance. Blood samples were
collected from the sows and were subjected to DNA tests to identify the presence of
specific genes affecting litter size and resistance against pre-weaning diarrhea, these genes
are: Estrogen Receptor Gene (ESR), Prolactin Receptor Gene (PRLR), and Leukemia
Inhibitory Factor Gene (LIF), these genes are the ones responsible in increasing the litter
size of sows. And 1, 2-Fucosyl Transferase gene (FUT1) for the disease resistance. Sow
individual performance records are also collected to see the relationship of these genes with
the actual performance of the sows. The summary of performance data from the farm is
given in Table 4.
Table 4. Farm 1 Performance record from year 2015 up to present.

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 Parameter 2015 2016 2017* Ideal**
Total Piglets Born 590 1121 351
Total Piglets Born/Litter 13.1 11.6 14.0 11.5
Total Piglets born Alive 538 1070 314
Piglets Born Alive/litter 11.9 11.0 12.6 10.9
Total Piglets Weaned 470 961 254
Piglets weaned/litter 10.4 9.9 10.2 10
Percentage Weaned, % 87.4 89.8 80.9 90
Weaned Sows 45 97 25
Ave weaning wt./piglet, kg. 5.9 5.1 5.2
Stillbirth, (%) 32 (5.4%) 32 (2.8%) 25, (7.1%) <7%
Mummified, (%) 20 (3.4%) 19 (1.7%) 12 (3.4) <1.5%
Deaths due to scouring 68 (12.6%) 95 (8.9%) 49 15.6%)
Pre-weaning Losses, % 12.6 8.9 15.6 10%
*2017 data is from Jan-Mar only
**Ideal values are based on Swine Stockmanship Standards.

There were twice as many farrowing and piglets born in 2016 relative to 2015 and 2017 as
well which was to be expected as 2017 is only partial record. Nevertheless, the average
number of piglets born alive per litter and the average number of piglets weaned per litter
for the three-year period were all above average with 2016 having the lower values.
However, the percentage of piglets weaned in 2015 and 2017 were lower than ideal due
almost exclusively to pre-weaning mortalities due to diarrhea. For 2017, pre-weaning
losses is already 15.6% and intervention or preventive action may be needed to lower the
losses. Affected litters from scouring increased from 32 in 2015 to 47 in 2016, in 2017 49
piglets have already died to scouring during the first quarter. Based on the submitted farm
data, the highest average weight of weaned piglets was on year 2015 with a value of 5.94kg,
but also has the highest number of mummified piglets of 20. In year 2016 weaned sows
doubled in value from 45 to 97 so as the number of piglets born from 590 to 1121 due to
addition of new sows to the farm, but with a substantial decrease of prenatal mortality from
8.81% down to 4.54%. Despite the percentage of mummified piglets higher than the ideal
which is 1.5% from 2015-2017, the average piglets born alive per litter is higher (11%
compared to the ideal which is 10.9%). The highest number of piglets weaned per litter
was in 2015 (10.4), but the highest number of weaned piglets was on 2016 (961). Despite
the high number of piglets weaned on 2016, there was a decrease on piglets weaned per
litter observed that may be linked to an increase in the number of piglets that died on
scouring.

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Genetic testing for fertility traits

Litter size
The genotype and allele frequencies are shown in Table 5. Based on the data, the frequency
of the favorable allele A and the genotype homozygous for the favorable allele (BB) for
ESR gene is much higher for this herd compared to the result from the previous project.
Given the high frequency, it would suggest that the source of genetics for the sow line of
this farm maybe selecting specifically for this gene. The frequency of favorable allele for
LIF gene was is also high however, these are mostly found in heterozygous genotypes.
Sires used could mostly be homozygous for the favorable allele with very small percentage
heterozygous. The frequency for the PRLR gene follows the same pattern as the other two
genes but on the reverse as there are less favorable alleles in the data set. The frequencies
obtained could be a correlated response to selection as different genes may affect the same
trait but their influence on specific measures of the same trait may vary. In this study, we
only looked at litter size, specifically, number of piglets born alive. Other measures of
fertility such as days to first post-partum service was not included.

Table 5. Genotype and allele frequencies for the three genes affecting fertility traits that
were tested.
GENOTYPE ALLELE
GEN AA AB BB A B
E
N (%) N (%) N (%) % %
ESR 37.0 22.7 70.0 42.9 56.0 34.4 44.2 55.8
PRLR 34 20.4 69 41.3 64 38.3 41.0 59.0
LIF 2 1.2 127 76.0 38 22.8 39.2 60.8
Favorable allele: ESR allele B, PRLR allele A, LIF allele B

The desirable genes affecting fertility (ESR, PRLR, and LIF) have a positive effect on litter
size of sows as shown in Table 6. Generally, the positive effect was more pronounced on
older sows with more parities with some sows having litter sizes as high as 14-16 piglets
per litter with the highest of 20, although some sows have a sudden drop on their litter size
in their 2nd parity. It appeared that sows carrying the favorable allele is more likely to stay
longer in the herd longer.

Table 6. Average number of piglets born alive per litter of sows carrying favorable alleles
of genes for fertility traits.
GENOTYPE
AA AB BB
Parity No. Overall Average N
Mean N Mean N Mean N
ESR
1 10.6 9 12.4 19 11.7 15 11.7 43
2 9.7 9 10.9 19 11.2 15 10.6 43
3 12.6 9 12.2 14 13.5 10 12.7 33
4 12.2 5 11.4 8 10.2 6 11.2 19

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 5 12.3 3 10.7 7 9.8 5 10.7 15
6 13.5 2 9.3 3 13.8 5 12.4 10
Average/genotype 11.4 37 11.5 70 11.7 56 11.5 163

LIF
1 13 1 11.8 32 11.3 12 11.7 45
2 6 1 11.0 32 10.0 12 10.6 45
3 13.3 24 11.1 9 12.7 33
4 10.9 15 12.3 4 11.2 19
5 10.3 14 17.0 1 10.7 15
6 12.4 10 12.4 10
Average/genotype 9.5 2 11.7 127 11.1 38 11.5 167

PRLR
1 10.2 9 11.6 17 12.5 19 11.7 45
2 11.4 9 10.1 17 10.8 19 10.6 45
3 13.0 6 12.5 13 12.8 14 12.7 33
4 9.3 4 10.8 9 13.2 6 11.2 19
5 11.7 3 10.4 8 10.8 4 10.7 15
6 12.3 3 12.2 5 13.0 2 12.4 10
Average/genotype 11.2 34 11.2 69 12.0 64 11.5 167
Favorable allele: ESR allele B, PRLR allele A, LIF allele B

The positive effect of genes on the litter size is best shown on older sows with a 2-3 piglets
increase per litter. Of the three desirable genes affecting litter size, sows carrying allele B
Leukemia Inhibitory Factor (LIF) gene best depicts the positive effect of genes on the litter
sizes (Table 7) and is further shown in Table 8 with both Total Number Born and Total
Born Alive piglets were highest in LIF followed only by ESR. But still both gene shows
the positive effect of animals having the favorable genes to those who do not.

Genes affecting litter size, despite its positive effects, are also known to negatively affect
the growth rate of the pigs, but luckily these types of genes have a very low heritability of
only 0.1. Based on the sow performance data provided, sows positive on markers have a
little to no negative impact on the average weaning weight of piglets as shown in Figures
3 & 4.

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Figure 3. Genes Affecting Litter Size on Average Weaning Weight. Desirable
Homozygous Genotypes

Figure 4. Genes Affecting Litter Size on Average Weaning Weight.

Heterozygous/Recessive Genotypes

Table 7. Sows with litter sizes of 12 and above that are sorted based on genotype per
individual genes.
ESR PRLR LIF
Parity
No. Genotype Genotype Genotype
AA AB BB AA AB BB AA AB BB
1st 4 13 8 2 10 13 1 18 6
2 nd 2 9 9 6 6 9 0 16 5
3rd 6 10 7 4 8 11 0 20 3
4 th 3 4 3 1 5 4 0 8 2
5th 2 3 1 1 3 2 0 5 1
6 th 1 0 3 1 2 1 0 4 0

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Table 8. Sows with litter sizes of 15 and above that are sorted based on genotype per
individual genes.
ESR PRLR LIF
Parity
No. Genotype Genotype Genotype
AA AB BB AA AB BB AA AB BB
1st 0 5 0 0 1 4 0 4 1
2nd 1 3 4 3 2 3 0 6 2
3rd 2 4 4 2 2 6 0 8 2
4th 1 1 1 0 0 3 0 2 1
5th 0 1 1 0 1 1 0 1 1
6th 1 0 2 1 1 1 0 3 0

Table 9. Total Number Born Piglets and Piglets Born Alive per Parity Grouped
according to sows genotype. A bold digit indicates desirable genotype for the specific
gene.
Genotype
Parity No. AA AB BB
TN TBA TN TBA TN TBA
ESR
1st 108 95 248 235 196 175
2nd 95 87 216 207 172 168
3rd 125 113 195 171 137 135
4th 63 61 97 91 61 61
5th 37 37 80 75 49 49
6th 34 27 31 28 78 69
PRLR
1st 104 92 211 197 258 237
2nd 110 103 179 171 213 205
3rd 80 78 176 162 201 179
4th 38 37 102 97 81 79
5th 35 35 88 83 43 43
6th 41 37 76 61 26 26
LIF
1st 15 13 418 378 140 135
2nd 6 6 371 353 125 120
3rd 0 0 334 319 123 100
4th 0 0 169 164 52 49
5th 0 0 147 144 19 17
6th 0 0 143 124 0 0

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Genetic testing for disease resistance

Escherichia coli (F18) resistance

Based on the results from the test regarding disease resistance specifically FUT1 for the
resistance to pre-weaning diarrhea, Figure 5 shows no animals have the genes that are
resistant to the disease, 60% of the sows in the population has the GG Susceptible genotype,
while the remaining 40% has the susceptible Heterozygous genotype. This explains the
high mortality rate of piglets due to lack of resistance to pre-weaning diarrhea. Table 10
further shows the total genotypic and allelic distribution of the FUT1 gene per breed in the
farm.

AA Resistant 0
AG Susceptible 18 AA Resistant
0%
GG Susceptible 27

AG Susceptible
40%
GG Susceptible
60%

Figure 5. FUT1 Genotypic Distribution on sows

Table 10. Genotype and allele frequency of FUT1.

GENOTYPE ALLELE
BREED AA AG GG A G
N (%) N (%) N (%) % %
Large White 0 0 82 41.41 116 58.58 20.7 79.29
Landrace 0 0 14 23.33 46 76.67 11.6 88.33

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Table 11. Comparison between genotypes of FUT1 showing average mortality rates per
year from pre-weaning diarrhea
GENOTYPE
AG GG
Year Parity No. % No. %
No. No.
N Weane Mort N Weane Mort
Mort. Mort.
d . d .
2015
1
1 5 11.6 0.6 4.51 4 8.9 2.5 40.3
1
2 5 10.8 0.6 4.52 4 10.6 1.4 15.0
3 3 12.3 1.3 10.47 4 13.0 1.0 8.0
2016
1
1 7 10.4 1.7 19.31 6 10.6 0.9 9.9
1
2 6 11.7 0.7 5.56 5 8.1 0.7 6.0
1
3 2 12.0 0.0 0.00 4 10.2 1.3 13.6
1
4 5 11.2 0.6 5.58 4 9.9 1.1 9.7
2017
2 1 12.0 0.0 0.00 1 3.0 0.0 0.0
3 5 12.6 2.2 17.56 2 12.5 1.0 8.0
Average/genotyp 3 9
e 9 11.5 1.0 9.25 4 9.8 1.3 14.9

Despite previous studies mentioning that both AG and GG genotypes are both susceptible
to pre-weaning diarrhea. AG genotypes, as shown in Table 11, has still lower mortalities
as compared to the GG genotypes, in which we can now assume that even having only an
allele from the resistant gene AA has already an impact on the resistance against the pre-
weaning diarrhea.

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