Genomic insight of sperm motility
Subodh KumarSenior Scientist
Division of Animal GeneticsIndian Veterinary Research Institute
Izatnagar (Bareilly) UP
1st Feb 2013
Introduction
Apr 12, 2023
2003 2007
Cattle 185.181 199.075
Buffalo 97.922 105.343Total 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%
Apr 12, 2023
Milk Production (Million Tonnes)
BAHS, 2010
Apr 12, 2023
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
Apr 12, 2023
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 Apr 12, 2023
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
Apr 12, 2023
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
Basic Sperm Structure
FLAGELLA
MITOCHONDRIA
ION CHANNELS
METABOLISM
AKAP4
CatSper I
SPAG6
SPAG11
CatSper II
CatSper IIICatSper 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 Xp11.2Mouse:XA1.6
Start : 49,842,146 bp End : 49,852,404 bpSize : 10,259 basesORF : 2565bpExons : 6Translation : 854 amino acid
AKAP4 – Map location in Human
AKAP4 location in Mouse
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 HumanSimilarity
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 : 22,674,406End : 22,746,545Genomic Size : 72,141ORF : 1524 bpExons : 11Aminoacids : 507
MAP LOCATION
Human 10p12.2Mouse 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
INTRONSPROMOTER
Orthologs for SPAG6 gene
OrganismHuman
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)
SPAG6 Expression
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 pterEnd : 7,308,602 bp from pterSize : 15,917 bases Exons : 8ORF : 327 nt Translation : 108 aa
Map location
Human : 8p23.1Cattle : 27q1.2Mouse : 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’ UTRINTRONS
EXONS
I II III IV V VI VII VIII
OrganismHuman
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
Start : 40,716,154 bp from pterEnd : 40,728,061 bp from pterSize : 11,908 basesExons : 11ORF : 1227 ntTranslation : 408 amino acid
MAP LOCATION
Human : 19q13.1Mouse : 7B1Cattle : 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
OrganismHuman
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 expression
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 LOCATIONHuman : 1q21Mouse : 3F1Cattle : 3
Start : 151,117,422 bp from pter
End : 151,124,147 bp from pter
Size : 6,726 basesExon : One ORF : 351 bp length Translation : 116 amino acid
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).
SMCP expression
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 AHuman: 11q13.1Cattle: 29
CatSper 1 gene summary
Start : 65,540,799 bp from pterEnd : 65,550,564 bp from pterSize : 9,766 basesORF : 2343 NucleotideProtein : 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’UTREXONS (12) 2443 bp
INTRONS
Orthologs for CatSper1 gene
OrganismHuman
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
Start : 41,707,993 bp from pterEnd : 41,728,338 bp from pterSize : 20,346 basesExons : 12ORF : 1593 nt Translation : 530 aa
Map location
Human : 15p15.3Cattle : 21Mouse : 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 ntINTRONS
Orthologs for CatSper2 gene
OrganismHuman
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)
CatSper3 gene summary
Start : 134,331,495 bp from pter
End : 134,375,291 bp from pter
Size : 43,797 bases
ORF : 1197 nt
Translation : 398 aa
Map location
Human : 5q31.1Mouse : 13B2
CatSper3 gene organisation
VIIIII VII VIII IV VII
5’UTR3’UTR
98 154 240 183 141 120 158 103
Exons Introns
Orthologs of CatSper3
OrganismHuman
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)
CatSper4 gene summary
MAP LOCATION
Human : 1p35.3Mouse : 4D3Rat : 5q36
Start : 26,389,706 bp from pterEnd : 26,401,620 bp from pterSize : 11,915 basesEXONS : 10 ORF : 1419 nt Protein : 472 amino acids
CatSper4 gene organisation
I II IV V VI VIII IX
213 144 102 98 121 134 175 212 166 54
5’UTR 3’UTR
Orthologs for CatSper4 gene
OrganismHuman
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 237bp282bp385bp299bp
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 bpCat1-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. : 150C
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 cyclesCyclic 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
S. No. Steps Temp. Time
1. Initial denaturation 95 °C 4min
2.35 cycles
Cyclic denaturation 94 °C 45 sec
Cyclic annealing 62.5°C 45 sec
Cyclic extension 72 °C 45 sec
3. Final extension 72 °C 10min
4. Storage 4°C 10min
73
SSCP patterns in Cat1 Exon2
AA (97)70.30 %, AB (16)18.10%, BB(25)11.60%
AA AA AB AA BB AB AA AA AA AB AA BB AB AA
74
PCR-Amplification for Catsper1 exon3
237bp
100bp ladder
S. No. Steps Temp. Time
1. Initial denaturation 95 °C 4min
2.35 cycles
Cyclic denaturation 94 °C 45 sec
Cyclic annealing 63°C 45 sec
Cyclic extension 72 °C 45 sec
3. Final extension 72 °C 10min
4. Storage 4°C 10min
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
PCR-Amplification for Catsper1 exon4
385 bp
100bp ladder
S. No. Steps Temp. Time
1. Initial denaturation 95 °C 4min
2. 35 cyclesCyclic
denaturation94 °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
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
PCR-Amplification for Catsper1 exon5
282bp
100bp ladder
S. No. Steps Temp. Time
1. Initial denaturation 95 °C 4min
2. 35 cyclesCyclic
denaturation94 °C 45 sec
Cyclic annealing 55°C 45 sec
Cyclic extension 72 °C 45 sec
3. Final extension 72 °C 10min
4. Storage 4°C 10min
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
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82
Haplotype analysis covering five exons of Catsper1 gene in crossbred bulls
Sl.No. Bull No.
Exon1 Exon2 Exon3 Exon4 Exon5 Haplotype
Pattern Pattern
Gen Pattern
Gen Pattern
Gen Pattern
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
3 89 ZZ AA G/G FF C/C JJ A/A MM C/C I-GC
4 402 ZZ AA G/G FF C/C JJ A/A MM C/C I-GC
5 969 ZZ BB A/A FF C/C JJ A/A MM C/C III-AC
6 992 ZZ AA G/G FF C/C JJ A/A MM C/C I-GC
7 1034 ZZ AA G/G FF C/C JJ A/A MM C/C I-GC
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
Total96 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
HaplotypeSeminal parameters
MM IPM PTM
I-GC 3.22a ± 0.10
67.66a
±1.4543.22a ±1.06
II-(G/A)T 3.47a ± 0.18
68.57a
± 2.4943.33a
±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.
90
Thank you