Small Ruminant Research 141 (2016) 127–134

Contents lists available at ScienceDirect

Small Ruminant Research

journal homepage: www.elsevier.com/locate/smallrumres

Interleukin-2 ((IL-2) gene polymorphism and association with heat

tolerance in Nigerian goats
A. Yakubu a,b,c,∗ , A.E. Salako c , M.D. Donato a,d , M.I. Takeet a,e , S.O. Peters f , M. Wheto g ,
M. Okpeku h,i , I.G. Imumorin a,∗∗
a
Animal Genetics and Genomics Laboratory, International Programs, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY 14853, USA
b
Department of Animal Science, Nasarawa State University, Lafia, Nigeria
c
Department of Animal Science, University of Ibadan, Ibadan, Nigeria
d
Laboratorio Genetica Molecular, IBB, Universidad de Oriente, Cumana, Venezuela
e
Department of Veterinary Microbiology and Parasitology, University of Agriculture, Abeokuta, Nigeria
f
Department of Animal Science, Berry College, GA 30249, USA
g
Department of Animal Breeding and Genetics, University of Agriculture, Abeokuta, Nigeria
h
Department of Livestock Production, Niger Delta University, Amassoma, Nigeria
i
State Key Laboratory of Genetic Resources and Evolution, Chinese Academy of Science (CAS), Kunming Institute of Zoology, Kunming, Yunnan, China

a r t i c l e i n f o a b s t r a c t

Article history: Interleukin-2 (IL-2) serves as a potent immuno-modulator, and plays a unique role in both the activation
Received 11 May 2016 and maintenance of immune responses and in lymphocyte development. Gene polymorphism in IL-2 gene
Received in revised form 16 July 2016 in three major Nigerian goat breeds [West African Dwarf (WAD), Red Sokoto (RS) and Sahel (SH)] was
Accepted 18 July 2016
analysed by restriction fragment length polymorphisms (RFLP). The restriction enzyme, DraI was utilized.
Available online 20 July 2016
The association between the polymorphic site and some heat tolerant traits were also investigated in a
total of 157 animals comprising 59 WAD, 69 RS, and 29 SH goats of both sexes. The IL-2 sequence displayed
Keywords:
a 98% nucleotide identity with the referenced Cahi-IL-2 AY603404.1 orthologous sequence in GenBank.
IL-2
Polymorphisms
The phylogenetic trees revealed largely species-wise clustering, while the RS and SH goats were closer
Heat stress compared to the WAD goats. DraI genotype frequencies were in Hardy-Weinberg Equilibrium (P < 0.05).
Phylogeny The expected heterozygosity (H), which is a measure of gene diversity in the goat populations, ranged from
Goats 0.26 to 0.28. The heterozygote (TA) had a comparative advantage over the homozygote (AA) in terms of
rectal temperature (39.21 ± 0.08 (TT) and 38.86 ± 0.13 (TA); P < 0.05). The SH goats also displayed greater
resistance to thermal stress compared to the RS and WAD goats. There were varying sex and interaction
effects on the thermal indices. The present findings may find application in the selection of superior
animals for resistance to heat stress as well as provide insight to questions about evolution, ecology and
conservation of Nigeria indigenous goats.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction expressed in the central nervous system of healthy organisms also

Cytokines have generally been discovered and characterized as
components of the peripheral immune system. Although far from
fully understood, the role of cytokines as immune-modulators in
the peripheral immune system is well documented (Dhama et al.,
2015). That several cytokines and their receptors are constitutively
∗ Corresponding author at: Department of Animal Science, Nasarawa State Uni-
versity, Lafia, Nigeria.
∗∗ Corresponding author.
E-mail addresses: abdulmojyak@gmail.com, abdul mojeedy@yahoo.com
(A. Yakubu), igi2@cornell.edu (I.G. Imumorin).
supports the notion that cytokines may have originally evolved
for functions other than signaling in the peripheral immune sys-
tem (Opp, 2005). Cytokines have been shown to play important
roles in different aspects of animal health and production (Marcos-
Carcavilla et al., 2007; Lühken et al., 2009; Szydlowski et al., 2010;
Darlay et al., 2011). Cytokines are mediators of information trans-
fer between cells, and in this way, they regulate physiological and
pathological mechanisms in the body (Turner et al., 2011). They are
potentially of use in diagnosis. IL-2 is the prototype member of a
family of cytokines that have pleiotropic actions in the immune sys-
tem (Lin and Leonard, 2003). It was discovered in 1976 as a growth
factor present in conditioned medium from phytohemagglutinin
(PHA)-stimulated normal human lymphocytes. This was able to

http://dx.doi.org/10.1016/j.smallrumres.2016.07.015
0921-4488/© 2016 Elsevier B.V. All rights reserved.
128 A. Yakubu et al. / Small Ruminant Research 141 (2016) 127–134
specifically support the growth of activated normal T lymphocytes
in-vitro and was therefore denoted as T-cell growth factor (TCGF).
IL-2 stimulates natural killer (NK) cells, and lymphocyte-activated
killer (LAK) cells. It shows strong B-cell growth factor activity
and can stimulate monocytic lineage cells (Pintaric et al., 2008;
Nagarajan et al., 2011). It also stimulates progesterone production
in granulose cells (Kumar and Esha, 2011). The insulin-dependent
diabetes susceptibility locus 3 (idd3) in the NOD mouse has been
shown to map to the IL-2 gene (Yamanouchi et al., 2007). Luhken
et al. (2005) reported the presence of transcription factor binding
sites in the second intron of the IL-2 gene of cattle. The SNPs in these
sites might influence the binding of transcription factors leading
to varied production patterns in different genotypes. Genotypes
arising from IL-2 polymorphism have been reported to have signif-
icant associations with milk production traits. This can also serve
as a major tool for combating disease susceptibility and improv-
ing overall productivity of livestock populations in the tropics by
selecting and breeding the most suitable genotypes (Prakash et al.,
2011).
The goat (Capra hircus, L.) represents one of the most important
farm animal species. It is reared in all continents with an estimated
world population of about 900 million of animals, out of which
55.1 million are reared in Nigeria (FAOSTAT, 2011). The Nigerian
goat is still genetically unimproved, and the pressure of modern
genetic improvement has increased the need to better understand
natural genetic variation in Nigerian goat breeds, as well as formu-
late germplasm conservation and improvement policies. Although
the three major breeds of goats in Nigeria namely, West African
Dwarf (WAD), Red Sokoto (RS) and the Sahel goats have been
characterized using morphological traits (Yakubu et al., 2011), pro-
tein polymorphism (Imumorin et al., 1999; Yakubu et al., 2014),
Fig. 1. Map of Nigeria showing sampling locations.
microsatellites (Okpeku et al., 2011) and major histocompatibity
complex DQB1 markers (Yakubu et al., 2013), there is no informa-
tion on genetic characterization involving interleukin genes.
Therefore, this study aimed at investigating molecular genetic
variation and phylogenetic diversity of IL-2 gene in Nigerian goat
breeds and the association of its SNPs with heat tolerance traits. The
molecular markers (SNPs) emanating from the association between
IL-2 allelic variations and heat tolerant traits in this study would lay
the ground work for appropriate molecular selection strategies for
sustainable production and effective management of tropical goats
especially in Nigeria, sub-saharan Africa characterized by high envi-
ronmental temperatures.
2. Materials and methods
2.1. Animals and blood collection
Blood samples were obtained from a total of 157 animals (about
2 years of age) comprising 59 West African Dwarf (WAD), 69
Red Sokoto (RS) and 29 Sahel (SH) goats of both sexes sampled
across various farms in Nigeria (Fig. 1). The ethical guidelines of
the International Council for Laboratory Animal Science and Cor-
nell University, Ithaca, NY, USA were strictly followed. Genomic
DNA was extracted from the collected blood samples using the
ZymoBeadTM Genomic DNA kit (Zymo Research Corp. Irvine, CA,
USA). The quantification of the DNA yield as well as the assessment
of its quality was done using Nanodrop ND-100 UV/Vis Spectropho-
tometer (Nanodrop Technologies, Inc., DE, USA).
 A. Yakubu et al. / Small Ruminant Research 141 (2016) 127–134 129

2.2. PCR amplification and sequencing of caprine IL-2 gene with their normal average values was used to derive HSI according
to the method of Oladimeji et al. (1996):
A primer pair for the amplification of the 462-bp of IL-2 gene
in 39 animals (13 per breed) for polymorphism identification was HSI = (RR/PR) × (NPR/NRR)
designed as described by Luhken et al. (2000). The forward primer where,RR = measured respiratory ratePR = measured pulse
was 5 -AAGAGT CAT CAG AAG AGG AAA-3 while the reverse was rateNPR = normal pulse rateNRR = normal respiratory rate.
5 -AAC CTT GGG CAT GTA GAA GT-3 . PCR amplifications were car-
ried out in a C1000TM Thermal Cycler (Bio-Rad, USA) with a total
2.5. Sequence and phylogenetic analysis
reaction volume of 20 ␮L containing 20–30 ng DNA, 10 pmol of each
primer in 10 ␮L Syd Lab PCR Premix (Syd Labs, Inc., Malden, USA)
Sequence alignments, translations and comparisons were
containing Taq DNA polymerase, dNTPs, MgCl2 , reaction buffer,
carried out using ClustalW. The BLAST algorithm was used
PCR stabilizer and enhancer at optimal concentrations. Thermo-
to search the NCBI GenBank (http://www.ncbi. nlm.nih.gov/)
cycling conditions consisted of 35 cycles of initial denaturation at
databases for homologous sequences. Nine Nigerian goat IL-2
94 ◦ C for 5 min, denaturation at 94 ◦ C for 30 s; primer annealing at
sequences (3 each of WAD, RS and SH goats) and pub-
56 ◦ C for 1 min 30 s, extension at 72 ◦ C for 1 min 30 s and a final
lished goat (U34274.1, AF535145.1 and EF056469.1), sheep
extension for 10 min at 72 ◦ C. PCR products were detected on 1.0%
(NM 001009415.1, AF287479.1, EF056466.1 and X60149.1,), cattle
agarose gel including 0.5 ␮g/ml of GelRed nucleic acid stain and
(U24226.1, BC153237.1 and EF056472.1) and swine (NM 213835.1;
scored using GENEMate Quanti-Marker 100 bp DNA ladder (Bio-
which serves as outgroup) IL-2 sequences were compiled.
Express, UT, USA). The PCR products were visualized after the gel
IL-2 sequences of Nigerian goats and those of published goat,
electrophoresis using ultra violet transilluminator (SpectrolineR TC
sheep and cattle sequences were used to obtain a phylogenetic
312 E) and photographed under UV light (Alpha imager). Sequenc-
tree applying the p-distance option of UPGMA of MEGA version 5
ing of the amplified fragments was carried out using the same PCR
(Tamura et al., 2011). The reliability of the branching was estimated
primer with the Applied Biosystems Automated 3730XL DNA Ana-
by bootstrap confidence values with 500 bootstrap replications.
lyzer using Big Dye Terminator chemistry and AmpliTaq-FS DNA
Similarly, Consensus sequence of each of Nigerian goat breeds and
Polymerase.
that of published caprine sequence were used to obtain a phyloge-
netic tree using the UPGMA method. All the IL-2 sequences were
2.3. SNP identification and restriction fragment length edited to a similar length of 462 bp.
polymorphism (RFLP) analysis
2.6. Statistical analysis
Single nucleotide polymorphisms (SNPs) were identified in IL-2
gene with the aid of CodonCode Aligner software and MEGA version Genotype and allele frequencies of DraI were determined by
5 (Tamura et al., 2011). In the RFLP analysis, 20 ␮L of concentrated direct counting as described by Falconer and Mackay (1996). Allele
PCR products were digested for 4 h at 37 ◦ C with DraI (Fermentas, and genotype frequencies of each gene in each goat population
USA) according to the manufacturers’ instructions. The restriction were tested for Hardy–Weinberg Equilibrium (HWE), and the dif-
fragments were resolved by 1.0% agarose gel electrophoresis and ferences between the observed and expected numbers of each allele
were visualized using ultra violet transilluminator (SpectrolineR TC and genotype were compared using a goodness-of-fit chi-square
312 E). (␹2 ) test. HWE was assumed for P > 0.05 using POPGENE Statistical
Software (Yeh et al., 1999). Heterozygosity (He) was estimated as
the expected proportion of heterozygotes under HWE. Data were
2.4. Physiological data collection and analysis
also analysed using the general linear model (GLM) of SAS (2010)
statistical software to test the fixed effects of SNP genotype, breed
Rectal temperature (RT, ◦ C), skin temperature (ST, ◦ C), respira-
and sex as well as their interactions on physiological indices (RT,
tory rate (RR, breaths per minute) and pulse rate (PR, beats per
ST, RR, PR and HSI). Means were separated using Duncan’s Multiple
minute) were measured early in the morning between 7:00 a.m.
Range Test (DMRT) at 95% confidence interval. The following linear
and 8:00 a.m. before sunrise. Repeated measurements for RT, ST, RR
model was employed:
and PR were carried out between 4:00 p.m. and 5:00 p.m. Thermo-
physiological measurements were taken on adult animals in the yijkl = ␮ + Gi +Bj + Sk + (GB)ij + (GS)ik + (BS)jk + (GBS)ijk + eijkl
months of November, February and March corresponding to the
period of peak environmental temperatures in Nigeria. Sampling
yijkl = individual observation
was done in such a way that each breed and sex was adequately
represented in each month. This was executed to avoid the element
of bias which might give erroneous results especially where there  = overall mean
existed too few animals in a particular breed and a very low number
of a particular sex. In this wise, 59 WAD (20 males and 39 females), Gi = fixed effect of ith SNP genotype (i = TT, TA)
69 RS (19 males and 50 females) and 29 SH goats (16 males and 13
females) were utilized. Unhealthy and pregnant animals were not Bj = fixed effect of jth breed (j = WAD, RS, SH)
sampled as these may influence thermo-physiological parameters.
RT was taken on each animal using a digital thermometer. The
sensory tip was disinfected and inserted into the rectum at the Sk = fixed effect of kth sex (k = male, female)
display of L◦ C by a thermometer (which indicated that the ther-
mometer is set for temperature reading). This was removed after (GB)ij = interaction effect of ith SNP genotype and jth breed
the sound of the alarm signal. The displayed body temperature was
then recorded. RR was determined by counting the number of flank
(GS)ik = interaction effect of ith SNP genotype and kth sex
movements per minute. PR was measured by placing the fingertips
on the femoral arteries of the hind limb for 1 min. Heat stress index
(HSI): the relationship between the measured RR and PR together (BS)jk = interaction effect of jth breed and kth sex
130 A. Yakubu et al. / Small Ruminant Research 141 (2016) 127–134
Fig. 2. A 462-fragment size of IL-2 gene in Nigerian goats.
(GBS)ijk = interaction effect of SNP genotype, breed and sex
eijkl = random error associated with each record (normally,
independently and identically distributed with zero mean and
constant variance).
3. Results
3.1. Amplification of IL-2 region
The 462-bp fragment (Fig. 2) of the Nigerian goat IL-2 gene
displayed a 98% nucleotide identity with the reference Cahi-IL-2
AY603404.1 orthologous sequence in GenBank.
Fig. 3. Phylogenetic tree derived from sequences of each of Nigerian goat breeds and those of published caprine, ovine and bovine IL-2 alleles using the p-distance model of
UPGMA method. Porcine-IL-2 allele was used as an out-group. Only bootstrap values higher than 50% are shown.
3.2. Phylogenetic trees from IL-2 sequences
The phylogenetic tree clearly revealed that clustering was
largely species-wise (Fig. 3) in IL-2 gene. The dendrogram con-
structed from the consensus nucleotide sequence data revealed
that the southern WAD goats are more closely related to the north-
ern RS goats compared to their SH counterparts (Fig. 4).
3.3. Gel image of product of restriction enzyme of IL-2 gene
Gel image of the DraI RFLP (IL-2 gene) of Nigerian goats is pre-
sented in Fig. 5. Two genotypes TT and TA were observed in Nigerian
goats.
3.4. DraI genotypes and gene frequencies in Nigerian goats
Using the reference sequence with accession no. AF535145, DraI
(T414A) caused a change from TTT (phenylalanine) to TTA (leucine).
Two alleles (T and A) and two genotypes (TT and TA) were observed
in DraI RFLP (Fig. 1; Table 1). There was no AA genotype. The TT and
TA genotype frequencies were 0.69 and 0.31 (WAD), 0.65 and 0.35
(RS) and 0.72 and 0.28 (SH). The respective T and A gene frequencies
were 0.85 and 0.15 (WAD), 0.83 and 0.17 (RS) and 0.86 and 0.14
(SH). The Chi-square (␹2 ) analysis showed no significant differences
(␹2 = 3.841; (P > 0.05) in the observed and the expected frequencies
of the two genotypes which is consistent with Hardy Weinberg
distribution. The heterozygosity values were 0.26, 0.28 and 0.24
for WAD, RS and SH goats, respectively.
3.5. Influence of IL-2 SNP genotypes on heat tolerant traits of
Nigerian goat breeds
The effects of DraI genotypes on heat tolerant traits of Nigerian
goat breeds are presented in Table 2. The heterozygote (TA) had a
comparative advantage over the homozygote (AA) in terms of rectal
temperature [39.21 ± 0.08 (TT) and 38.86 ± 0.13 (TA); P < 0.05].
 A. Yakubu et al. / Small Ruminant Research 141 (2016) 127–134 131

Fig. 4. Phylogenetic tree of Nigerian goat and published caprine IL-2 sequences.

Table 1
Distribution of DraI (IL-2) genotypes and gene frequencies in Nigerian goats.

Breed Male Female Total Genotype Frequency Gene frequency Chi-square H

TT TA AA T A

WAD 20 39 59 41 (0.69) 18 (0.31) 0 (0.00) 0.85 0.15 1.97ns 0.26

RS 19 50 69 45 (0.65) 24 (0.35) 0 (0.00) 0.83 0.17 3.18ns 0.28
SH 16 13 29 21 (0.72) 8 (0.28) 0 (0.00) 0.86 0.14 0.73ns 0.24

Chi-square (␹2 ) (P < 0.05) = 3.841; ns not significant (P > 0.05).

H = Heterozygosity (gene diversity index).

Table 2
Effect of DraI SNP genotypes on heat tolerant traits of Nigerian goat breeds.

SNP Pulse rate Respiratory rate Skin Temperature Rectal Temperature Heat stress index

TT 102.33 ± 2.93 45.98 ± 1.46 38.49 ± 0.12 39.21 ± 0.08a

2.04 ± 0.09
TA 104.14 ± 4.99 43.68 ± 2.49 38.31 ± 0.20 38.86 ± 0.13b 1.77 ± 0.15

Means along the same column with different superscripts are significantly different (P < 0.05).

3.6. Heat tolerant traits of Nigerian goat breeds as affected by was also observed in terms of heat stress index [1.28 ± 0.15 (SH),
breed 1.86 ± 0.15 (RS) and 2.58 ± 0.09 (WAD); P < 0.05].

Breed variation in the thermoregulation and heat tolerance
of Nigerian goats was observed (Table 3). While the WAD goats
seemed better in pulse rate [86.36 ± 4.14 (WAD), 109.47 ± 6.41 (SH)
and 113.88 ± 4.13 (RS); P < 0.05], the SH goats appeared superior in
terms of respiratory rate (35.03 ± 3.20 (SH), 48.47 ± 2.07 (RS) and
35.03 ± 3.20 (SH); P < 0.05]. The comparative advantage of SH goats
3.7. Heat tolerant traits of Nigerian goat breeds as affected by sex
Sex had significant effect (P < 0.05) on pulse rate [97.01 ± 4.07
(male) and 109.47 ± 4.12 (female)] and rectal temperature
[38.93 ± 0.11 (male) and 39.15 ± 0.11 (female)] (Table 4).
3.8. Effects of SNP and breed interaction on heat tolerant traits of
Nigerian goats

The analysis of variance revealed varying interaction effects on

the heat-tolerance traits (Table 5). SNP and breed interaction under
(DraI) had significant effect (P < 0.05) on RR and HSI (Table 6). In
respect of RR, Genotype TT in WAD had higher value compared to
RS and SH goats. However, the effect of TA on RR was similar in all
the three goat breeds. As regards HSI, Genotype TT in WAD was also
higher compared to RS and SH goats. Genotype TA in SH goats was
lower in HSI value than their WAD counterparts.

3.9. Effects of SNP, breed and sex interaction on heat tolerant

traits of Nigerian goats

Genotype TT in WAD male was higher (P < 0.05) in RR value than

RS and SH goats (Table 7). However, TA in WAD and SH male goats
was lower in RR value than RS goats. Genotype TT in WAD females
had higher RR value compared to their RS and SH counterparts.
However, TA in SH females was lower in RR value compared to
WAD goats.

4. Discussion

The breeding objective in most livestock species is to increase

profit by improving performance efficiency. One way to reach
this objective is to improve the animals’ health, for example,
Fig. 5. DraI RFLP of IL-2 locus in Nigerian goat a (single band) and b (double through the implementation of appropriate management meth-
bands) = genotypes TT and TA, respectively. ods. A more sustainable method consists in taking advantage, by
132 A. Yakubu et al. / Small Ruminant Research 141 (2016) 127–134

Table 3
Effect of breed on heat tolerant traits of Nigerian goat breeds.

Breed Pulse rate Respiratory rate Skin Temperature Rectal Temperature Heat stress index

WAD 86.36 ± 4.14 b

50.99 ± 2.07 a
38.54 ± 0.17 39.00 ± 0.11 2.58 ± 0.09a
RS 113.88 ± 4.13a 48.47 ± 2.07a 38.30 ± 0.17 39.18 ± 0.11 1.86 ± 0.15b
SH 109.47 ± 6.41a 35.03 ± 3.20b 38.35 ± 0.26 38.95 ± 0.17 1.28 ± 0.15c

Means along the same column with different superscripts are significantly different (P < 0.05).

Table 4
Effect of sex on heat tolerant traits of Nigerian goat breeds.

Sex Pulse rate Respiratory rate Skin Temperature Rectal Temperature Heat stress index

Male 97.01 ± 4.07b 45.16 ± 2.03 38.23 ± 0.16 38.93 ± 0.11a 1.98 ± 0.12
Female 109.47 ± 4.12a 44.50 ± 2.06 38.57 ± 0.16 39.15 ± 0.11b 1.83 ± 0.12

Means along the same column with different superscripts are significantly different (P < 0.05).

Table 5
Analysis of variance showing the interaction effects of DraI SNP genotype, breed and sex on heat tolerant traits of Nigerian goats.

Source of variation Mean squares and level of significance

DF PR RR ST RT HSI
SNP × Breed 2 1186.51 ns 1070.85** 1.00ns 0.60ns 2.83*
SNP × Breed × Sex 2 14.64ns 751.51* 0.69ns 0.37ns 0.96ns
Residual 145 766.31 191.50 1.22 0.54 0.65

DF = degree of freedom, PR = pulse rate, RR = respiratory rate, ST = skin temperature, RT = rectal temperature, HSI = heat stress index.
ns
Non-significant.
*
Significant at P < 0.05.
**
Significant at P < 0.01.

Table 6
Interaction effect of SNP and Breed (DraI) on heat tolerant traits of Nigerian goats.

Breed SNP Parameters

PR RR ST RT HSI

WAD TT 78.89 ± 4.56 57.39 ± 2.28a 38.72 ± 0.18 39.30 ± 0.12 2.97 ± 0.13a
TA 93.83 ± 6.92 44.58 ± 3.46a 38.36 ± 0.28 38.69 ± 0.18 2.19 ± 0.20a
RS TT 115.09 ± 4.46 45.14 ± 2.23b 38.21 ± 0.18 39.25 ± 0.12 1.75 ± 0.13b
TA 112.67 ± 6.97 51.80 ± 3.48a 38.40 ± 0.28 39.10 ± 0.18 1.97 ± 0.20ab
SH TT 113.01 ± 6.05 35.40 ± 3.02c 38.54 ± 0.25 39.09 ± 0.16 1.40 ± 0.18b
TA 105.92 ± 11.30 34.67 ± 5.65a 38.17 ± 0.45 38.80 ± 0.30 1.16 ± 0.33b

Means along the same column with different superscripts are significantly different (P < 0.01, RR; P < 0.05, HSI).

Table 7
Interaction effect of SNP, breed and sex (DraI) on heat tolerant traits of Nigerian goats.

Sex SNP Breed Parameters

PR RR ST RT HSI

Male TT WAD 74.93 ± 7.40 58.71 ± 3.698a 38.39 ± 0.30 39.27 ± 0.20 3.121 ± 0.22
RS 108.57 ± 7.40 44.27 ± 3.698b 38.22 ± 0.30 39.32 ± 0.20 1.884 ± 0.22
SH 93.30 ± 8.75 33.70 ± 4.376b 38.41 ± 0.35 38.92 ± 0.23 1.620 ± 0.25
TA WAD 97.50 ± 11.30 37.33 ± 5.650b 38.00 ± 0.45 38.57 ± 0.30 1.828 ± 0.33
RS 112.40 ± 12.38 57.60 ± 6.189a 38.68 ± 0.50 39.26 ± 0.32 2.180 ± 0.36
SH 95.33 ± 11.30 39.33 ± 5.650b 37.68 ± 0.45 38.25 ± 0.30 1.235 ± 0.33

Female TT WAD 82.85 ± 5.327 56.07 ± 2.663a 39.06 ± 0.21 39.33 ± 0.14 2.823 ± 0.16
RS 121.61 ± 4.972 46.00 ± 2.485b 38.20 ± 0.20 39.18 ± 0.13 1.620 ± 0.14
SH 132.73 ± 8.347 37.09 ± 4.172b 38.66 ± 0.33 39.26 ± 0.22 1.178 ± 0.24
TA WAD 90.17 ± 7.99 51.83 ± 3.995a 38.72 ± 0.32 38.82 ± 0.21 2.545 ± 0.23
RS 112.95 ± 6.35 46.00 ± 3.175ab 38.11 ± 0.25 38.94 ± 0.17 1.764 ± 0.18
SH 116.50 ± 19.57 30.00 ± 9.785b 38.65 ± 0.78 39.35 ± 0.52 1.075 ± 0.57

Means along the same column with different superscripts are significantly different (P < 0.05).

selective breeding, of the within-breed variation that exists in the
mechanisms of defenses against infectious pathogens (Jovanovic
and Vicovac, 2009; Detilleux, 2011). Success against microbial
pathogens in immunopathological terms is a delicate balance
between mounting a potent innate and adaptive immune response
and limiting tissue destruction due to effector mechanisms (De
Silva et al., 2011). IL-2 stimulates the proliferation and differen-
tiation of T cells and the activation of monocytes and macrophages,
thereby playing an important role in regulating the immune
response in ruminants (Zhao et al., 2011).
The genetic relationships of IL-2 gene in Nigerian goats and
published caprine, ovine and bovine sequences showed by the
phylogenetic tree were in accordance with the speciation of these
species in the evolution history. The pattern of the phylogeny simi-
larity and differentiation in WAD (found mainly in the southern part
of the country), RS and SH (predominate in the northern Nigeria)
 A. Yakubu et al. / Small Ruminant Research 141 (2016) 127–134
goats suggests a direct relationship between the extent of adap-
tive divergence and geographical separation. As cytokine function
tends to be conserved across taxa, incorporating measurements of
these important molecules into studies of ecological immunology
will offer insights into many different host-parasite interactions
(Hawley and Altizer, 2011). Therefore, the high level of conser-
vation observed in the IL-2 sequences of livestock species in the
present study may help in developing a common therapeutic agent
targeted to all the major farm animals (Sreekumar et al., 2002).
IL-2 polymorphism therefore represents an important source of
variation between individuals in immune function and pathogen
resistance. This variation may, in part, be maintained by exposure
to multiple pathogen species, such that a genotype conferring resis-
tance to one pathogen species may confer increased susceptibility
to a second (Turner et al., 2011). These associations may represent
an example of antagonistic pleiotropy, which has been proposed as
a mechanism by which genetic diversity may be maintained within
a population (Rose, 1982; Roff et al., 2005).
The much greater frequency of the T allele may be due to natu-
ral selection favouring animals carrying the T allele, or genotypes
having the T allele may be artificially selected along with other
characters favoured by artificial selection. The heterozygous geno-
type arising from the polymorphic site in the present study might
prove useful. This is in consideration of the pivotal role of IL-2
gene during the occurrence of diseases and its effect on animal
productivity (Prakash et al., 2011) as well as characterization and
evolutionary study. A major impetus for studying the cytokines of
farm animals is to evolve alternate disease management strate-
gies (Lowenthal et al., 2000). In a preliminary study, Bressani et al.
(2011) reported a SNP identified within the intron 2 of IL-2 gene
which showed association with resistance against gastrointesti-
nal infection by nematodes. Alongside their direct immunological
roles, cytokines also have a wide-range of non-immune physio-
logical effects including influences on thermoregulation, appetite
and fatigue (Corwin, 2000). It is therefore likely that an alteration
in the action of cytokines will have a more significant effect on
host pathology than simply its impact on infectious disease sus-
ceptibility. The significance of the physiological role played by IL-2
was consolidated by Burchill et al. (2007) who implicated IL-2 in
the differentiation and homeostasis of so-called natural Tregs that
develop in the thymus of animals.
It has been reported that exosomes from heat-stressed 3LL Lewis
lung tumor cells effectively elicit systemic antitumor immunity,
and that IL-2 gene-modified tumor cells were more efficient in pro-
moting dendritic cells (DC) maturation (Wang et al., 2014). This
further attest to the role played by IL-2 gene in immune function.
Under thermal stress, a number of physiological and behavioural
responses vary in intensity and duration in relation to the animal’s
genetic make-up, gender and environmental factors (Hughes et al.,
2013). If genetic selection for superior responses to heat stress is
possible, then it ought to be possible to breed robust and easily
managed genotypes that might be able to adapt to a wide range
of environmental conditions whilst expressing a high production
potential (Hough et al., 2013). In this wise, genotype TA found in this
study may be a good candidate for possible resistance to heat stress
in the tropics. A better picture of genetic contributions to suscepti-
bility to heat tolerance and autoimmune conditions would certainly
help improve our understanding of the pathophysiology of live-
stock diseases which could assist in making appropriate breeding,
selection, evolutionary, ecological and conservation decisions.
The study revealed that the SH goats had a comparative advan-
tage in terms of heat tolerance compared to the WAD and RS goats.
This could also be exploited in selection and breeding policies
geared towards increased productivity. Sex effect did not follow a
definite pattern, as there were varying responses to thermal indices
by both sexes.
133
Under SNP and breed interaction, SH and RS goats with genotype
TT stand to be selected because they appeared to be less stressed in
terms of RR. Genotype TT and TA in SH and RS goats offer superior
advantage in terms of HSI. The interactions of breed, sex and SNP
genotype on RR suggest the sensitivity of the expressed forms of IL-
2 genotypes to breed and sex differences. Selection tends to favour
genotypes TT and TA in both sexes of SH goats, whereas genotype
TT in RS males and TA in RS females appeared to be less stressed. TA
in WAD male goats also appeared to be favourable. There is dearth
of information on the interaction effect of IL-2 genotypes and heat
tolerance traits in caprine species.
5. Conclusion
The study revealed that Nigeria goats clustered well with pub-
lished caprine nucleotide sequences at the IL-2 locus. RS and SH
goats were also closer at this locus compared to the WAD goats.
The heterogygote TA had the potential to resist heat stress com-
pared to the homogygous TT. The heat tolerant ability of SH goats
also appeared better than that of WAD and RS goats. The polymor-
phic site obtained in IL-2 gene of Nigerian goats when genotyped in
a large population may be exploited in making appropriate breed-
ing, selection, evolutionary, ecological and conservation decisions.
It will also be interesting for future studies to investigate the asso-
ciation of the obtained IL-2 SNP with susceptibility or resistance to
the prevailing livestock diseases in the country.
Conflict of interest
The authors declare that there is no conflict of interest.
Contribution of authors
AY, IGI and AES designed the work. AY, MW and MO carried out
the field work. AY, MDD, MIT, MW, SOP and IGI did the laboratory
work and statistical analysis. AY and AES wrote the manuscript. All
authors read and approved the final manuscript.
Acknowledgements
We thank the College of Agriculture & Life Sciences and Mario
Einaudi Center for International Studies of Cornell University,
Ithaca, NY for financial support for this project. Additional sup-
port from the Nigerian Government Tertiary Educational Trust Fund
fellowship to AY and MIT is gratefully acknowledged.
References
Bressani, F.A., Tizioto, P.C., Meirelles, S.L., Malago Junior, W., Giglioti, R., Ibelli,
A.M.G., Gromboni, J.G.G., Carrilho, E., Zaros, L.G., da Silva Vieira, L., Correia de
Almeida Regitano, L., 2011. Identification of a SNP in the gene IL2 and its
association with resistance against gastrointestinal infection by nematodes in
goat. J. Anim. Sci. 89 (E-Suppl. 1), 280–281, J. Dairy Sci. Vol. 94, E-Suppl. 1
(abstract).
Burchill, M.A., Yang, J., Vang, K.B., Farrar, M.A., 2007. Interleukin-2 receptor
signaling in regulatory T cell development and homeostasis. Immunol. Lett.
114 (1), 1–8.
Corwin, E.J., 2000. Understanding cytokines: part I: physiology and mechanism of
action. Biol. Res. Nurs. 2, 30–40.
Darlay, R.J., McCarthy, A.J., Illot, N.E., Smith, J.E., Shaw, M.-A., 2011. Novel
polymorphisms in ovine immune response genes and their association with
abortion. Anim. Genet. 42, 535–543.
De Silva, K., Begg, D., Whittington, R., 2011. The interleukin 10 response in ovine
Johne’s disease. Vet. Immunol. Immunopathol. 139, 10–16.
Detilleux, J.C., 2011. Effectiveness analysis of resistance and tolerance to infection.
Genet. Sel. Evol. 43, 9, http://dx.doi.org/10.1186/1297-9686-43-9.
Dhama, K., Saminathan, M., Jacob, S.S., Singh, M., Karthik, K., Tiwari Amarpal, A.R.,
Sunkara, L.T., Malik, Y.S., Singh, R.J., 2015. Effect of immunomodulation and
immunomodulatory agents on health with some bioactive principles, modes of
action and potent biomedical applications. Int. J. Pharmacol. 11, 253–290.
134 A. Yakubu et al. / Small Ruminant Research 141 (2016) 127–134
FAOSTAT, 2011. Food and Agriculture Organization of the United Nations.
(available at http://faostat.fao.org/default.aspx). (accessed 11.08.15.).
Falconer, D.S., Mackay, T.F.C., 1996. Introduction to Quantitative Genetics, 4th
edition. Longman, Harlow, England.
Hawley, D.M., Altizer, S.M., 2011. Disease ecology meets ecological immunology:
understanding the links between organismal immunity and infection
dynamics in natural populations. Funct. Ecol. 25, 48–60.
Hough, D., Swart, P., Cloete, S., 2013. Exploration of the
hypothalamic-pituitary-adrenal axis to improve animal welfare by means of
genetic selection: lessons from the South African Merino. Animals 3, 442–474.
Hughes, H.D., Carroll, J.A., Sanchez, N.C.B., Richeson, J.T., 2013. Natural variations in
the stress and acute phase responses of cattle. Innate Immun. 20, 888–896.
Imumorin, I.G., Ologun, A.G., Oyeyemi, M.O., 1999. Preliminary observations on
effects of haemoglobin genotype and estimate of genetic distance at the Hb
locus in West African Dwarf and Red Sokoto goats. Trop. J. Anim. Sci. 1,
1–9.
Jovanovic, M., Vicovac, L., 2009. Interleukin-6 stimulates cell migration, invasion
and integrin expression in HTR-8/svneo cell line. Placenta 30, 320–328.
Kumar, S.R., Esha, G., 2011. Effect of interleukin −2 on steroidogenesis of goat
ovary. J. Immunol. Immunopathol. 13, 20–24.
Lühken, G., Wagner, H.W., Seichter, D., Hecht, W., Erhardt, G., 2009. Genetic
characterization of a sheep-dwarf goat hybrid. Cytogenet. Genome Res. 125,
158–161.
Lin, J.-X., Leonard, W.J., 2003. Interleukin-2. In: Thomson, Angus W., Lotze, Michael
T. (Eds.), The Cytokine Handbook. , 4th edition. Elsevier B.V, Netherlands, pp.
167–199.
Lowenthal, J.W., Lambrecht, B., Vandenberg, T.P., Andrew, M.E., Strom, A.D.G.,
Bean, A.G.D., 2000. Avian cytokines—the natural approach to therapeutics. Dev.
Comp. Immunol. 24, 355–365.
Luhken, G., Stamm, I., Menge, C., Erhardt, G., 2005. Functional analysis of a single
nucleotide polymorphism in a potential binding site for GATA transcription
factors in ovine IL-2 gene. Vet. Immunol. Immunopathol. 107, 51–56.
Marcos-Carcavilla, A., Calvo, J.H., González, C., Moazami-Goudarzi, K., Laurent, P.,
Bertaud, M., Hayes, H., Alves, M.E.F., Serrano, M., 2007. Short communication:
IL-1 family members as possible candidate genes affecting economically
important traits in cattle. Span. J. Agric. Res. 5, 38–42.
Nagarajan, U.M., Sikes, J., Prantner, D., Andrews Jr., C.W., Frazer, L., Goodwin, A.,
Snowden, J.N., Darville, T., 2011. MyD88 deficiency leads to decreased NK cell
gamma interferon production and T cell recruitment during Chlamydia
muridarum genital tract infection, but a predominant Th1 response and
enhanced monocytic inflammation are associated with infection resolution.
Infect. Immun. 79, 486–498.
Okpeku, M., Ozoje, M.O., Adebambo, O.A., Agaviezor, B.O., O’Neill, M.J., Imumorin,
I.G., 2011. Preliminary analysis of microsatellite-based genetic diversity of
goats in southern Nigeria. Anim. Genet. Res. 49, 33–41.
Oladimeji, B.S., Osinowo, O.A., Alawa, J.P., Hambolu, J.O., 1996. Estimation of
average values for pulse rate, respiratory rate and rectal temperature and
development of a heat stress index for adult Yankasa sheep. Bull. Anim. Health
Prod. Afr. 44, 105–107.
Opp, M.R., 2005. Cytokines and sleep. Sleep Med. Rev. 9, 355–364.
Pintaric, M., Gerner, W., Saalmuller, A., 2008. Synergistic effects of IL-2, IL-12 and
IL-18 on cytolytic activity: perforin expression and IFN-gamma production of
porcine natural killer cells. Vet. Immunol. Immunopathol. 121, 68–82.
Prakash, V., Bhattacharya, T.K., Jyotsana, B., Pandey, O.P., 2011. Molecular cloning,
characterization, polymorphism, and association study of the interleukin-2
gene in Indian crossbred cattle. Biochem. Genet. 49, 638–644.
Roff, D.A., Benedikt, H., Brian, K.H., 2005. Variation and Life-History Evolution.
Variation. Academic Press, Burlington, pp. 333–357.
Rose, M.R., 1982. Antagonistic pleiotropy, dominance, and genetic variation.
Heredity 48, 63–78.
SAS, 2010. Statistical Analysis System. Base SAS® 9.2 Procedures Guide: Statistical
Procedures, 3rd edition. Cary, NC: SAS Institute Inc. SAS Inst., Inc., Cary, NC,
USA.
Sreekumar, E., Premraj, A., Saravanakumar, M., Rasool, T.J., 2002. Buffalo (Bubalus
bubalis) interleukin-2: sequence analysis revealed high nucleotide and amino
acid identity with interleukin-2 of cattle and other ruminants. Eur. J.
Immunogenet. 21, 341–345.
Szydlowski, M., Buszka, A., Mackowski, M., Lechniak, D., Switonski, M., 2010.
Polymorphism of genes encoding cytokines IL6 and TNF is associated with pig
fatness. Livest. Sci. 136, 150–156.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA 5
molecular evolutionary genetics analysis using maximum likelihood,
evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28,
2731–2739.
Turner, A.K., Begon, M., Jackson, J.A., Bradley, J.E., Paterson, S., 2011. Genetic
diversity in cytokines associated with immune variation and resistance to
multiple pathogens in a natural rodent population. PLoS Genet. 7, e1002343,
http://dx.doi.org/10.1371/journal.pgen.1002343.
Wang, J., Wang, L., Lin, Z., Tao, L., Chen, M., 2014. More efficient induction of
antitumor T cell immunity by exoxomes from CD40L gene-modified lung
tumor cells. Mol. Med. Rep. 9, 125–131.
Yakubu, A., Salako, A.E., Imumorin, I.G., 2011. Comparative multivariate analysis of
biometric traits of West African Dwarf and Red Sokoto goats. Trop. Anim.
Health Prod. 43, 561–566.
Yakubu, A., Salako, A.E., De Donato, M., Takeet, M.I., Peters, S.O., Adefenwa, M.A.,
Okpeku, M., Wheto, M., Agaviezor, B.O., Sanni, T.M., Ajayi, O.O., Onasanya, G.O.,
Ekundayo, O.J., Ilori, B.M., Amusan, S.A., Imumorin, I.G., 2013. Genetic diversity
in Exon 2 at the major histocompatibility complex DQB1 locus in Nigerian
indigenous goats. Biochem. Genet. 51, 954–966.
Yakubu, A., Abimiku, H.K., Musa-Azara, I.S., Barde, R.E., Raji, A.O., 2014. Preliminary
investigation of haemoglobin polymorphism and association with
morphometric traits in West African Dwarf goats in north central Nigeria.
Mljekarstvo 64, 57–63.
Yamanouchi, J., Rainbow, D., Serra, P., Howlett, S., Hunter, K., Garner, V.E.,
Gonzalez-Munoz, A., Clark, J., Veijola, R., Cubbon, R., Chen, S.L., Rosa, R.,
Cumiskey, A.M., Serreze, D.V., Gregory, S., Rogers, J., Lyons, P.A., Healy, B.,
Smink, L.J., Todd, J.A., Peterson, L.B., Wicker, L.S., Santamaria, P., 2007.
Interleukin-2 gene variation impairs regulatory T cell function and causes
autoimmunity. Nat. Genet. 39, 329–337.
Yeh, F.C., Yang, R.C., Boyle, T., Ye, Z.H., Mao, J.X., 1999. POPGENE, the user Friendly
Shareware for population Genetic Analysis. Molecular Biology and
Biotechnology Center, University of Alberta.
Zhao, K., He, W., Gao, W., Lu, H., Han, T., Li, J., Zhang, X., Zhang, Z., Wang, G., Gaoli
Su, G., Zhao, Z., Song, D., Gao, F., 2011. Orf virus DNA vaccines expressing ORFV
011 and ORFV 059 chimeric protein enhances immunogenicity. Virol. J. 8, 562,
http://www.virologyj.com/content/8/1/562.