Genomic insight into genes regulating sperm motility

Genomic insight of sperm motility
Subodh Kumar
Senior Scientist
Division of Animal Genetics
Indian Veterinary Research Institute
Izatnagar (Bareilly) UP
1st
Feb 2013

Introduction
May 12, 2014
2003 2007
Cattle 185.181 199.075
Buffalo 97.922 105.343
Total
Bovine 283.446 304.765BAHS, 2010
Livestock Population in Million

Cattle Population
DAHD 2007
1997
(Million)
2003
(Million)
2007
(Million)
Growth%
(03-07)
Cross Bred 20.09 24.68 33.06 33.92
Indigenous 178.78 160.04 166.6 3.43
10.1%
89.9%
13.1%
86.4%
15.7%
83.6%
May 12, 2014

Milk Production (Million Tonnes)
BAHS, 2010May 12, 2014

Milk Production (MT)
BAHS, 2010
% of Cattle contribution
CB
46.98
IND
53.02
IND
81
CB
19
Indigenous Cross Bred
Population (Million) 52.13 12.5
Milk production (MT) 25.357 22.468
Crossbred Contribution
% of Milch cattle Population
May 12, 2014

Quality of crossbred semen as compared to exotic and zebu
counterparts in the same environment
Parameter B.
indicus
B.
taurus
B.Indicus x
B. taurus
References
Initial motility(%) 61.00 59.00
79.41
82.19
51.00
67.49
68.13
Singh and Pangawkar (1990)
Shrivastava and Kumar (2006)
Chacon et al.,(1999)
Head + mid piece
Abnormalities (%)
9.4% 15.1 19.0 Chacon et al.,(1999)
Tail Abnormalities +
cytoplasmic droplets(%)
14.0 13.7 18.0
Bull disqualifying rate(%) 54
48
Tyagi et al., (2006)
Chacon et al.,1999
Mass activity 4.00 3.06 Shrivastava and Kumar (2006)
SPD-CMPT (mm) 45.06 39.94
HOST(%) 49.38 42.06
Post Thaw Motility (%) 40.31 28.13 May 12, 2014

∗ Higher diseases incidence
∗ Requires intense managemental conditions
∗ Non-availability of superior crossbred germ plasms
(Mathew et al., 1982)
∗ Poor quality semen
(Rao and Rao, 1991)
∗ Poor freezability of semen and cryo-injuries
(Ghosh et al., 2007)
∗ Poor motility and viability of sperms
(Dhanju et al., 2006)
∗ High percentage of dead and abnormal sperms
(Ghosh et al., 2007)
Problems in crossbreds
May 12, 2014

Introduction
 Sperm motility - a complex process
 Parameters of semen quality
 Root cause of asthenzoospermia –
poorly understood
 Understanding of genetic mechanism
 Most knowledge based on human and
laboratory animals
 Sperm motility genes are highly conserved

Sperm Motility - Classification
 Morphological basis:
* Non-motile sperm
* Motile sperm
@ Progressively motile sperm
@ Non-progressively motile sperms
* Total motility
 Physiological basis:
* Activated motility
* Hyperactivated motility

 Freshly ejaculated sperm
 Flagellum generates a symmetrical lower
amplitude wave
 Move in a straight line in non viscous
media - seminal plasma or semen
extender
Activated motility

Hyperactivated motility
 Present in sperm after reaching Oviduct of
female reproductive tract
 Flagellar beats asymmetric and of higher
amplitude
 Move in circular or figure 8 trajectory
 Helps to penetrate egg vestment
 Seen in association with onset of
capacitation but are not dependent on each
other

FLAGELLA
MITOCHONDRIA
ION CHANNELS
METABOLISM
AKAP4
CatSper I
SPAG6
SPAG11
CatSper II
CatSper III
CatSper IV
GAPDS
SMCP
Candidate genes for Sperm Motility

 Encodes a member of the AKAP family.
 Alternative splicing of this gene results in two transcript
variants encoding different isoforms.
 Nearly half of the protein in fibrous sheaths isolated
from mouse sperm is AKAP4.
 This protein and two others, AKAP3 and AKAP-80, have
anchoring sites for cAMP-dependent protein kinase and
helps the cAMP/PKA signaling pathway
 Targeted disruption- causes defects in sperm flagellum
and motility.
AKAP4 (A Kinase Anchor Protein 4)

Map Location
Human Xp11.2
Mouse:XA1.6
Start : 49,842,146 bp
End : 49,852,404 bp
Size : 10,259 bases
ORF : 2565bp
Exons : 6
Translation : 854 amino acid
AKAP4 – Map location in Human

Organization of AKAP4
I II III IV V
156 2133 102 51 96 27
5’UTR 3’UTR
EXONS (6) 2565 bp
INTRONS
PROMOTER

Organism Human
Similarity
Dog
(Canis familiaris)
88.44(n)
84.02(a)
Chimpanzee
(Pan troglodytes)
98.84(n)
98.4(a)
Cow
(Bos taurus)
83.87(n)
78.7(a)
Rat
(Rattus norvegicus)
82.84(n)
78.65(a)
Mouse
(Mus musculus)
82.98(n)
79.27(a)
Orthologos for AKAP4 Gene

Expression pattern of AKAP4 Gene

SPAG6 (Sperm Associated Antigen6)
 Encodes an axonemal protein containing eight armadillo repeats
 Expressed in Male reproductive tissues particularly Epididymis
and Testis tissues
 SPAG6-deficient males were infertile.
motility defects,
morphologically abnormal (loss of the sperm head)
disorganization of flagellar structures,
 Important for the maintenance of the axonemal central
apparatus and structural integrity of mature sperm.
 Essential for sperm flagellar motility

Start : 22,674,406
End : 22,746,545
Genomic Size : 72,141
ORF : 1524 bp
Exons : 11
Aminoacids : 507
MAP LOCATION
Human 10p12.2
Mouse 16A3
Cattle 13
SPAG6 Gene summary

Organization of SPAG6
II III IV V VI VII IXVIII X
205 191183 173166 152 14524 95 69116
5’UTR
3’UTR
EXONS (11) 1524 bp
INTRONS
PROMOTER

Orthologs for SPAG6 gene
Organism
Human
Similarity
Dog
(Canis familiaris)
92.93(n)
96.27(a)
Cow
(Bos taurus)
91.94(n)
96.27(a)
Rat
(Rattus norvegicus)
87.42(n)
95.85(a)
Mouse
(Mus musculus)
87.57(n)
96.25(a)
Chicken
(Gallus gallus)
82.25(n)
85.8(a)

 Encodes androgen-dependent, epididymis -specific
secretory proteins. The specific functions not been
determined.
 Single gene derived from two ancestrally independent
β-defensin genes joined by read-through transcription
 Some isoforms contain regions similar to beta-
defensins.
 Rat epididymal cells or human colonic epithelial cells
transfected with rat SPAG11 could induce sperm
motility in immotile immature sperm. ( Zhou et al.
2004)
 Important for the acquisition of sperm motility and the
initiation of sperm maturation.
SPAG 11 (Sperm Associated Antigen 11)

SPAG11- gene summary
Start : 7,292,686 bp from pter
End : 7,308,602 bp from pter
Size : 15,917 bases
Exons : 8
ORF : 327 nt
Translation : 108 aa
Map location
Human : 8p23.1
Cattle : 27q1.2
Mouse : 8A1.1

Genomic organization of SPAG11
P1
A A A
98 153 151 107 76 259 104 279
PROMOTER
3’UTR
A- Poly adenylation sites
5’ UTR
INTRONS
EXONS
I II III IV V VI VII VIII

Organism
Human
Similarity
Rat
(Rattus norvegicus)
71.17(n)
62.16(a)
Mouse
(Mus musculus)
72.07(n)
61.26(a)
Orthology of SPAG11

 Encodes a protein belongs to GAPDS family of enzymes
that play an important role in carbohydrate metabolism.
 Functions in a NAD-dependent manner to remove hydrogen
and add phosphate to glyceraldehyde 3-phosphate to form
1,3-diphosphoglycerate.
 During spermiogenesis, play an important role in regulating
the switch between different energy-producing pathways,
and it is required for sperm motility and male fertility
GAPDS (Glyceraldehyde 3, phosphate Dehydrogenase
Sperm specific

GAPDS gene summary
Size : 11,908 bases
Exons : 11
ORF : 1227 nt
MAP LOCATION
Human : 19q13.1
Mouse : 7B1
Cattle : 18

Organization of GAPDS
I II VIII IXIII IV V VI VII X XI
67 178 97 107 91 119 82 152 163 98 73
5’UTR 3’UTR
EXONS (1227 bp)
INTRONS

Orthologs for GAPDS gene
Organism
Human
Similarity
Dog
(Canis familiaris)
84.49(n)
86.07(a)
Chimpanzee
(Pan troglodytes)
99.18(n)
98.77(a)
Cow
(Bos taurus)
85.57(n)
86.84(a)
Rat
(Rattus norvegicus)
79.49(n)
83.33(a)
Mouse
(Mus musculus)
79.41(n)
82.6(a)

GAPDS Protein
 68% identical with somatic cell GAPD.
 GAPDs has a 72-amino acid proline-rich segment at the amino terminal
end that is not present in somatic cell GAPD.
 Exists in sperms as the tetrameric molecule bound to the fibrous sheath of
the flagellum
 Cysteine residues (C21, C94, and C150) are specific for the sperm
isoenzyme
 Residue C21 involved in the formation of the disulfide bond between the
N-terminal domain of GAPDs and fibrous sheath proteins.
 Localised in the principal piece of the flagellum

 Encoded protein localised in Cytoplasm,
Mitochondrion membrane.
 Becomes associated with the mitochondrial outer
membranes at spermatogenesis
Function:
 Organization and stabilization of the helical structure
of the sperm & mitochondrial sheath
 Absence is associated with male infertility,
 Reduced sperm motility in female reproductive tract
 Inability to penetrate the oocyte zona pellucida
SMCP (Mitochondria Associated Cystein Sperm
rich Protein)

SMCP Gene summary
MAP LOCATION
Human : 1q21
Mouse : 3F1
Cattle : 3
Size : 6,726 bases
Exon : One
ORF : 351 bp length

SMCP
 The 5’ & 3’ UTR are more conserved (71%) than the coding
sequences (59%).
 The open reading frame encodes a 116-amino acid protein and lacks
the UGA codons.
 The MCSP gene in human, baboon, and bovine is more conserved
than its counterparts in mouse and rat.
 Expression is restricted to haploid spermatids in humans (Aho et al.
1996).
 The 5’ UTR of mouse, rat, and human SMCP also has a predicted
stem loop that is involved in regulation of SMCP expression.
(Hawthorne et al. 2006).

Discovery of a New Protein/Gene
• A new ion channel protein discovered (Clapham 2001).
• This was found to be expressed in testis (Ren et al., 2001).
• Expressed on the principal piece of sperm tail.
• Normal sperm (having this protein) showed faster progressive
movements.
• Those lacking it (gene disrupted), swim with 1/3rd
speed and move
more randomly.
(Clapham and Garbers, 2005)

More about the New Protein/Gene
• Mutants were 100% ineffective at impregnating females though they
displayed normal mating behavior.
• CASA = mutant sperm’s main motility parameters of path velocity,
progressive velocity and track speed were impaired significantly.
• Most notable, mutant sperm lacked the vigourous beating and bending
of the tail region.
• Thus disrupting this gene resulted in marked reduction in sperm motility
(Clapham and Garbers,
2005)

This gene/protein was called as
(Cation channel of Sperm)
CatSper is an Voltage gated Calcium selective ion channel
protein that plays a central role in sperm motility.
This newly discovered protein ‘controls flow of Calcium ions in to tail of
sperm’ which cause fibre like contractile proteins (motor proteins) to
produce forceful lashings of sperm tail.

Other researches on CatSper
• In human, subfertile men with defficient sperm cell motility had
significantly reduced (3.5 times) expression of CatSper (Nikpoor et al.,
2004).
• Sperm with Null CatSper (CatSper -/-
) were not able ascend the oviduct
whereas sperm from heterozygote (CatSper +/-
) were able to (Suarez et
al., 2005).
• Tiny electric currents generated by Ca+2
moving into sperm have also
been recorded which were called as CatSper dependent current (iCatSper
)
and shown to be an alkaline potentiated, voltage activated, calcium
selective channel (Kirichok et al., 2006).
• CatSper are highly specialized flageller protein and could be expressed
in HEK 293 cell line (Quill et al., 2007).

CatSper Characterization
• The mouse CatSper gene is predicated to have a primary
structures of 686 amino acids with six putative transmembrane
domains (S1-S6) and the pore region (P) which exists between
S5 and S6 (Jin et al., 2005).
• Out of six transmembrane domains, the fourth is a voltage
sensor (Jhang et al., 2006).
• Alternative forms of CatSper and conserved pore region (T X D
x W) motif reported in human (Asadi et al., 2006).

CatSper channel
 5 different genes encoding CatSper
1,2,3 ,4 & β
 Expressed in the plasma membrane
of principal piece of sperm tail
 Catsper protein is a single 6 TM
spanning repeat – closest to Cav
channels
 S4 segment acts as a voltage
sensor, abundance of histidine and
arginine residues

Heterotetramerization of CatSper channel

 CatSpers 1,2,3 & 4 may interact directly or indirectly and form a
functional tetramer
 CatSper1 and CatSper2 can associate with and modulate the
function of the Ca(v)3.3 channel, which might be important in
the regulation of sperm function.
 Controls calcium entry to mediate the hyperactivated motility,
 CatSper3 - has role in acrosome reaction and male fertility
 Catsper3 and Catsper4 knockout male mice were completely
infertile due to a quick loss of motility and a lack of
hyperactivated motility under capacitating conditions
 CatSper is therefore implicated as a potential target to explore
the molecular mechanisms of male infertility.
CatSper genes -Function

MAP Location
Mouse: 19 A
Human: 11q13.1
Cattle: 29
CatSper 1 gene summary
Size : 9,766 bases
ORF : 2343 Nucleotide
Protein : 780 aminoacid

CatSper 1 gene organisation
VIIV XIII XIX XII
27 115 76 61 73 64 144 92 148 114 213 1216
5’UTR 3’UTR
EXONS (12) 2443 bp
INTRONS

Orthologs for CatSper1 gene
Organism
Human
Similarity
Dog
(Canis familiaris)
82.98(n)
77.67(a)
Cow
(Bos taurus)
75.06(n)
62.88(a)
Rat
(Rattus norvegicus)
63.84(n)
53.94(a)
Mouse
(Mus musculus)
67.48(n)
57.67(a)

CatSper2 gene summary
Size : 20,346 bases
Exons : 12
ORF : 1593 nt
Map location
Human : 15p15.3
Cattle : 21
Mouse : 2E5

CatSper2 gene organisation
I II V VII XIIII VI VIII XIIXIX
32 165 218 57 100 179 125 156 173 69 174 145
5’UTR 3’ UTR
EXONS (12) 1593 nt
INTRONS

Organism
Human
Similarity
Dog
(Canis familiaris)
84.91(n)
79.36(a)
Chimpanzee
(Pan troglodytes)
99.37(n)
98.86(a)
Cow
(Bos taurus)
82.32(n)
76.52(a)
Rat
(Rattus norvegicus)
74.86(n)
70.06(a)
Mouse
(Mus musculus)
74.49(n)
69.47(a)

Size : 43,797 bases
ORF : 1197 nt
Map location
Human : 5q31.1
Mouse : 13B2

VIIIII VII VIII IV VII
5’UTR
3’UTR
98 154 240 183 141 120 158 103
Exons Introns

Orthologs of CatSper3
Organism
Human
Similarity
Dog
(Canis familiaris)
82.89(n)
76.06(a)
Chimpanzee
(Pan troglodytes)
99.66(n)
99.25(a)
Cow
(Bos taurus)
82.65(n)
75.89(a)
Rat
(Rattus norvegicus)
73.72(n)
67.83(a)
Mouse
(Mus musculus)
74.43(n)
66.67(a)

MAP LOCATION
Human : 1p35.3
Mouse : 4D3
Rat : 5q36
Size : 11,915 bases
EXONS : 10
ORF : 1419 nt
Protein : 472 amino acids

I II IV V VI VIII IX
213 144 102 98 121 134 175 212 166 54
5’UTR 3’UTR

Organism
Human
Similarity
Dog
(Canis familiaris)
82.76(n)
77.12(a)
Chimpanzee
(Pan troglodytes)
99.64(n)
99.78(a)
Cow
(Bos taurus)
83.88(n)
78.51(a)
Rat
(Rattus norvegicus)
79.15(n)
76.85(a)
Mouse
(Mus musculus)
77.78(n)
72.9(a)

LACK OF LITERATURE ON
ANY CATTLE (Bos spp)

Project executed in our lab
Title : Identification of SNPs in CatSper gene and their
association with sperm motility in cattle

65
Diagram of Exon1-5 for Catsper1 gene on cattle
E-5E-3E-2 E-4E-1 E-15
10 kb
376bp 237bp
282bp385bp299bp

66
Primers Designed (NC_007330) for SSCP analysis of
CatSper1 Gene in Cattle
Sl No Primer Name Primer Sequence Primer Length
(mer)
Product Length
(bp)
1. Cat1-E1F 5'AGTGGAAGCGCACAGTCCTA3' 20 mer 299 bp
Cat1-E1R 5'AGGGATGGACCCTAATGGAG3' 20 mer
2. Cat1-E2F 5'GGCCCATGTGTTAAGCTTTC 3' 20 mer 376 bp
Cat1-E2R 5'ATCCTGGGAAAGGGATGTG 3' 19 mer
3. Cat1-E3F 5'CAGAAGGCCTACCTCCATGA3' 20 mer 237 bp
Cat1-E3R 5'AAGACCGCTGGACGAGAATA3' 20 mer
4.
Cat1-E4F 5'GGGGAGTACCGTCATGGAAG3' 20 mer
385 bp
Cat1-E4R 5'GACTACACCAGCAGGGGAGA3' 20 mer
5.
Cat1E5F 5'CCTTTCTGGCCCCCTTACA3' 19 mer
282 bpCat1E5R 5'ACCAACATCAACGGCCTTCTCTAC3
'
24 mer

67
Optimized Conditions for PCR-SSCP Analysis
 Gel Conc. : 12%
 Time : 100bp/hr.
 Temp. : 150
C
 Voltage : 350 Volts
 Band patterns were resolved by Silver staining of
the gel (Bassam et al., 1991)

68
• Identification of SSCP band patterns
• Sequencing of SSCP patterns
• SNP identification by Nucleotide sequence analysis using MEGALIGN module
of DNA Star (Lasergene , USA) software.
• Estimation of Gene and Genotype frequency
Falconer and Mackey (2009)
• Haplotyping for SNPs data of each bull
• Anova using SAS programme (GLM, SAS 9.2) for ascertaining effect of
haplotype and its association with seminal parameters
• Statistical Model:
Yij = µ+Hi+eij
Where Hi is the effect of ith haplotype
µ = mean, eij =error effect
SNPs Identification and Association Study

69
Cattle Genomic DNA isolation
Conditions: Agarose gel 0.8%, 50 Volts for 1hr.
OD Ratio at 280/260: 1.6 to 1.9
Working DNA Concentration: 50-100ng/µl

70
PCR-Amplification for Catsper1 exon1
299bp
100bp ladder
S. No. Steps Temp. Time
1. Initial denaturation 95 °C 4min
2.
35 cycles
Cyclic denaturation 94 °C 45 sec
Cyclic annealing 64°C 45 sec
Cyclic extension 72 °C 45 sec
3. Final extension 72 °C 10min
4. Storage 4°C 10min

71
SSCP pattern (monomorphic) in Catsper1 Exon1 (299bp)
Pattern frequency (zz)=100%
zz zz zz zz zz zz zz zz zz zz zz zz zz zz
zz

72
PCR-Amplification for CatSper1 exon2
376bp
100bp ladder
2.
35 cycles
Cyclic annealing 62.5°C 45 sec

73
SSCP patterns in Cat1 Exon2
AA (97)70.30 %, AB (16)18.10%, BB(25)11.60%
AA AA AB AA BB AB AAAA AA AB AA BB AB AA

74
237bp
100bp ladder
2.
35 cycles

75
SSCP patterns in CatSper1 Exon3
DD DD EE FF EE GG GG HH
DD(58)42.03%, EE(34)24.64%, FF(25)18.12%, GG(9)6.52%, HH(12)8.69%

76
385 bp
100bp ladder
2. 35 cycles
Cyclic
denaturation
94 °C 45 sec

77
SSCP patterns Cat1 Exon 4
II X JJ JJ KK KK JJ LL
II(4)2.89%, JJ(101)73.19 %, KK(10)7.25%, LL(23)16.67%

78
282bp
100bp ladder
2. 35 cycles
Cyclic
denaturation
94 °C 45 sec

79
SSCP Patterns Cat1 Exon5
MM MM MN MN MN MN MN NN X NN NN NN NN NN
MM(21)15.22% MN(55)39.85% NN(62)44.93%

80
Total 10 novel SNPs identified in CatSper1 gene in cattle
Fragment
1
Fragment 2 Fragment 3 Fragment 4 Fragment 5
NO SNP T C,
199*(exon2)
C G
37(Intro1)
A G,
52
(Intron3)
G C
32 (exon5)
Val TO Leu
G A, 272
(exon2)
Gly TO Ser
C T, 94
(exon3)
Thr TO Met
A G,
160
(exon4)
Thr TO Ala
T G,
165
(Intron3)
T C,
345
(Intron4)
G
A,188
(Intron3)
* Silent mutation

81
Table: Fragment wise Gene and Genotype frequency for catsper1 gene in cattle
Fragment No. Genotype Frequency % Allele Frequency %
Fragment 1 ZZ (138) 100 Z 100
Fragment 2 AA (97) 70.30 A 79.35
AB (16) 18.10 B 20.65
BB(25) 11.60
Total 3 (138) 100 2 100
Fragment 3 DD(58) 42.03 D 42.03
EE(34) 24.64 E 24.64
FF(25) 18.12 F 18.12
GG(9) 6.52 G 6.52
HH(12) 8.69 H 8.69
Total 5 (138) 100 5 100
Fragment 4 II(4) 2.89 I 2.89
JJ(101) 73.19 J 73.19
KK(10) 7.25 K 7.25
LL(23) 16.67 L 16.67
Total 4 (138) 100 4 100
Fragment 5 MM(21) 15.22 M 35.14
MN(55) 39.85 N 64.86
NN(62) 44.93
Total 3 (138) 100 2 100
*Figuresinparenthesisindicatingnumberofanimals

82
Haplotype analysis covering five exons of Catsper1 gene
in crossbred bulls
Sl.No. Bull
No.
Exon1 Exon2 Exon3 Exon4 Exon5 Haplotype
Pattern Patte
rn
Gen Patte
rn
Gen Patter
n
Gen Patter
n
Gen
1 67 ZZ AA G/G FF C/C JJ A/A MM C/C I-GC
2 70 ZZ AB G/A DD T/T JJ A/A MM C/C II-(G/A)T
5 969 ZZ BB A/A FF C/C JJ A/A MM C/C III-AC

83
Seminal Parameters Evaluation
Bull No. N Vol.
(ml)
Conc
(mill/ml)
MM
(0-5)
IPM
(%)
PTM
(%)
(1) 70 V 21 3.95 ± 0.23 534.66 ± 52.10 3.47 ± 0.14 68.57 ± 2.41 43.33 ± 1.63
(2) 89 V 12 3.65 ± 0.28 533.91 ± 50.08 3.41 ± 0.19 72.91 ± 3.28 45.83 ± 2.20
(3) 1034 M1 18 4.17 ± 0.23 805.77 ± 49.63 3.94 ± 0.20 73.88 ± 2.27 48.88 ± 1.96
(4) 992 M1 12 4.05 ± 0.27 655.66 ± 47.62 3.41 ± 0.28 69.58 ± 3.45 45.83 ± 2.02
(5) 402 V 11 2.90 ± 0.33 671.54 ± 29.97 2.36 ± 0.15 61.36 ± 2.53 37.27 ± 1.56
(6) 969 V 14 4.63 ± 0.29 382.71 ± 39.87 1.21 ± 0.11 27.14 ± 2.26 12.50 ± 1.36
(7) 67 V 9 4.22 ± 0.27 566.66 ± 25.70 2.33 ± 0.16 53.33 ± 2.04 32.22 ± 2.22

84
ANOVA for Mass Motility
Source DF Sum of
Squares
Mean Square F
Value
Pr > F
Haplotype 2 52.5557429 26.2778715 35.07** <.0001
Error 94 70.4339478 0.7492973
Corrected
Total
96 122.989690

85
ANOVA for Initial Progressive Motility
Source DF Sum of
Squares
Mean
Square
F
Value
Pr >
F
Haplotype 2 19904.24545 9952.12273 75.88** <.0001
Error 94 12328.74424 131.15685
Corrected
Total
96 32232.98969

86
ANOVA for Post Thaw Motility
Source DF Sum of
Squares
Mean
Square
F
Value
Pr > F
Haplotype 2 11329.65442 5664.82721 79.97** <.0001
Error 94 6659.00538 70.84048
Corrected
Total
96 17988.65979

87
Association of bull fertility traits with
possible haplotypes
Haplotype
Seminal parameters
MM IPM PTM
I-GC 3.22a
± 0.10
67.66a
±1.45
43.22a
±1.06
II-(G/A)T 3.47a
± 0.18
68.57a
± 2.49
43.33a
±1.83
III-AC 1.21b
± 0.23
27.14b
± 3.06
12.50b
± 2.24

88
CONCLUSIONSCONCLUSIONS
 Five exons of catsper1 gene were PCR amplified in
cattle
 A total of 15 SSCP patterns were found
 10 novel SNPs were identified
 3 haplotypes in bulls
 Haplotype 1 and 2 significantly contributed to high
sperm motility

89
FUTURE PROSPECTS
 The association of Identified haplotypes with sperm
quality traits still need to be validated with large
number of bulls and that could be ultimately used for
Marker Assisted Selection (MAS).
 Genetic Effects of the identified DNA alteration at the
genomic level need to be confirmed at the
transcriptional or translational level.

Genomic insight into genes regulating sperm motility

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