Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
225
EVALUATION OF SEMEN SEX RATIO IN COOLED AND FROZEN
SEMEN STRAWS BY REAL-TIME PCR
Baqer Ja’fer. Hassan*; Souhayla Oneeis Hussain*; Mohammed Sh. Jebur**
*Department of Surgery and Obestetrics, College of Veterinary Medicine,Baghdad University.
** Department of Medical Analysis, College of Health and Medical Technology
Key word: Sex ratio, ZFX, SRY.
ABSTRACT
The study was demonstrated on quantitative evaluation of SRY and ZFX gene of (120) semen
straws from 10 Holliston bull (60) straws in cooling state 5C° and 60 straws after deep freezing
in liquid nitrogen) . The samples collected from the Artificial Insemination Center of Abo-
Ghreeb / Baghdad . All samples were sent to the laboratory for DNA extraction using (Qiamp
DNA extraction Kit) and primer design then testing in real time PCR. The results showed there
were highly significant variation in the sex ratio of cooled and frozen semen straws which varied
between (35%-59% in ZFX , 37%-58% in SRY and 40%-69% in ZFX , 30%-53% in SRY for
frozen and cooled semen respectively ) . The study conclude that it was easy and possible to
detect the quantity of sex ratio for each bull through using real time PCR . The freezing process
of semen could cause decrease in the percentages of SRY (minimum 30%) and increase in the
percentage of ZFX (maximum 69%) .
INTRIDUCTION
Many of developed countries are trying to pay attention to livestook, through that and for good
dairy industry female are more desirable at . It would be more profitable for dairy farmers to use
sexed sorting semen to produce daughters from genetically goal (1). Sex sorting of bull
spermatozoa has applications for genetic improvment in dairy industry with great economic
advantages and preselection for females will reduce the losses in both income and genetic
progress get by the producer (2).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
226
In artificial insemination industry, it is possible to collect, extend, package and ship a billions of
sperms cells from one male that will be used to breed thousands of females (3) . This allows for
the dispersal of superior genetic material through the these country (4) . Since the sperm cell
determining the sex of the offspring, semen sorted by its ability to produce males or females
would be very marketable(5) . Many techniques in past focused on the physical separation of Xand
Y- bearing spermatozoa, these methods were developed based on motility, DNA content
,surface charge, sperm surface antigenic determinants and the majority of these methods had non
disirable effects on semen quality (6). Other methods of separation had also been reported (7),
which were density gradient, electorophoresis, albumin centrifugation and sedimintation. All
these methods were not fully successful except flow cytometric cell sorting, in other hand a new
technique of quantification real time-Polymerase chain reaction (qRT-PCR) (8) has used to
determine the sex ratio of individual ejaculates of bull semen with an accuracy of 99% (9) depend
on the sex determination region of Y-chromosome gene(SRY) and ZFX gene (Zinc like finger of
X-chromosome gene)(10). The study has designed to investigate the effect of cooling and freezing
of semen on the bull's semen sex ratio depending on the percentages of sex determination region
of (SRY gene)and (ZFX gene).
MATERIAL AND METHODS
Ten (10) Holstein bulls located in Artificial Insemination Center, Baghdad-Abo Ghreeb, were
used for semen collection. Ages of these bulls ranging between (2-5year), and these were kept
under healthy environment and good management along the period of collection .Duration of the
study were (5 month) from 25-1-2016 to 25-6-2016 . The semen samples had collected by
artificial vagina then sent to the laboratory for routine semen tests(semen characteristics). Bad
semen (less than 49%) was discard, while the good semen (more than 50%) was acceptable for
the study and diluted by using egg yolk seminal (11)
The first group of samples collected at the cooling steps and the other group after 2 days post
deep freezing with liquid nitrogen (-196ºC) (Diagram :1) from the same ejaculate, each bull
alone .
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Diagram (1):The semen samples and experimental design.
PRIMER DESIGN: The present study was used primers that reported by (9) to determine the
sperm sex ratio by using of qRT-PCR which are forward (5-CCA CGT CAA GCG ACC CAT-
3) and reverse (5-AGA GCC ACC TTT CGT CG-3), which were used to amplify a 66 bp
fragment of SRY gene-y chromosome linking (12). The primershad designed for ZFX gene ( Xchromosome)
(5-GTT GTG TTA GTT TCT GCT GTA CAA TAA AGT G-3) and the reverse
(5-GAT GGC AGG TGA GGG TAG GA-3) for amplify 96 bp of DNA fragments (13) .
DNA EXTRACTION: Each one of the (120) samples placed in Eppendorf tubes at 37ºC water
path. Twentymicroliter of proteinase K had added, then 20 μl of ADTA and 10μl lyses buffer
solution also were added to each semen samples after mixed together and incubated for more
than 15 hours (overnight). The purification procedure of DNA used in this study based on spin
columns procedure which centrifuged at (500 cycle/minute) for 10 minute to collect the sperm
10 Bulls
120 straws
60 cooled straws 60 frozen straws
SRY gene ZFX gene SRY gene ZFX gene
RT-PCR RT-PCR
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
228
pellets (14). The samples were washed 4 times with 2.9% sodium citrate solution and incubated at
56 C for (30) minutes (15) then removing of cell depress by centrifugation with 1000 cycle/minute
for 5 minute. After that DNAs extract had purified using (QIAamp, DNA Mini Kit, Cat. No.
51304, QIAGE, GmbH, D. 40724 Hilden)
Real time PCR: After DNA extraction and purification, all samples had tested in real time PCR
(Sacycler% real time PCR system, 5x RUO, Italy)as the program below:
Step TemperatureºC Time min
1 Pre-denaturation 95:0 14:35
2 Denaturation and
Annealing
95:0 00:25
3 Extension 95:0 00:15
4 Melting curve 60:0 - 90:0 01:00 - 00:15
Table (1): Real time PCR program.
STATICAL ANALYSIS: The Statistical Analysis System (18) program was used to effect of
difference factors in study parameters. Chi-square test also had used for significant comparing
between percentages and Least Significant Difference (LSD) test.Through that the study was
estimating the correlation coefficient between characters of current study (9)
RESULTS AND DESCUSION
The results showed there were highly significant variation in the mean ratio of cooled and
frozen semen straws (Table:2). According to table-2the minimum mean percentage of SRY after
six weeks collection of semen and after cooling of semen straws were37% in bull number (41).
So that mean it was tend to produce females more than males because the mean percentage of
ZFX in the semen of this bull was more than 59%(percentage of Y-chromosome is less than
percentage of X-chromosome). While the maximum mean percentages of SRY gene in cooled
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
229
semen straws was 59% in bull number (17769) ,which mean it was tend to produce males more
than females because of this mean percentage of ZFX gene in same bull semen not exceed 35%
(mean percentage of Y-chromosome is more than X-chromosome) the result agreed with (16) .
The result after freezing show highly significant increase the ratio of ZFX (mean 56.5 %) and
decrease of SRY (mean 42.5%), the result was differenced with (16) when he found the ratio of
ZFX was (mean 50.3%) while the SRY was (49.6%) and even differenced with (9) who found the
semen sex ratio of ZFX (mean 54.7%) this variation in sex ratio may result from either using of
diluents or affected by semen processing and packaging in straws (the packaging process of
semen straws didn’t depend on the semen motility or activity but it is works according to
injection by special syringe with special system designed to kept in cooled condition) (17).
Bulls
Mean ± SE
After cooling After freezing
ZFX (X%) SRY (Y%) ZFX (X%) SRY (Y%)
4 48.83 ±9.51 51.17 ±9.51 47.67 ±1.20 52.33 ±1.20
57 55.67 ±8.48 39.67 ±7.91 56.17 ±7.61 43.83 ±7.61
145 58.50 ±8.15 41.17 ±7.98 66.17 ±5.04 34.00 ±5.09
41 59.40 ±4.95 37.00 ±1.67 69.20 ±2.95 30.80 ±2.96
109 47.50 ±2.78 54.67 ±1.90 62.50 ±3.58 37.50 ±3.58
528 37.83 ±2.79 56.00 ±4.08 59.17 ±2.66 40.83 ±2.66
409 47.33 ±1.45 52.67 ±1.45 55.00 ±1.52 45.00 ±1.52
17769 35.17 ±3.10 59.17 ±4.32 51.50 ±2.39 45.83 ±1.64
17768 58.75 ±2.65 44.00 ±5.36 58.25 ±3.31 41.75 ±3.30
451 42.00 ±4.51 58.00 ±4.50 40.33 ±1.20 53.67 ±6.43
LSD
value
18.736 * 18.596 * 12.386N * 12.663 *
* (P<0.05).
Table (2): Effects of bulls in % X and Y after cooling and freezing.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
230
At cooling steps , the results showed there were significant increase in ratio of ZFX in the
first two weeks (55.9% , 56.25% respectively ) (table : 3) and increasing of SRY in the third
week (57.28%) (figure :1) , this results were apposite with (16) when he found an increase in the
ratio of ZFX in the third and sixth weeks (53.8% , 58.8% respectively) , and increase in the ratio
of SRY in the first , 4th ,5th , weeks (53.4% , 54.2% , 54.4% respectively) . This variation in the
semen ratio may skewed from individual ejaculates of semen (9)
The frozen semen straws (figure:2) showed maximum increase in the ratio of ZFX in the second
week (62.12%) while the third week showed maximum increasing of SRY ratio (46.14%) (table
:3)
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Weeks
Mean ± SE
After cooling After freezing
ZFX (X%) SRY (Y%) ZFX (X%) SRY (Y%)
Week 1 55.90 ±6.17 44.10 ±6.17 58.30 ±4.28 39.90 ±3.83
Week 2 56.25 ±7.20 45.00 ±7.22 62.12 ±6.81 37.87 ±6.81
Week 3 43.00 ±4.96 57.28 ±4.71 54.00 ±2.91 46.14 ±2.87
Week 4 36.50 ±3.00 50.12 ±4.88 57.25 ±3.52 40.75 ±2.78
Week 5 49.10 ±3.35 50.90 ±3.35 55.00 ±2.21 45.00 ±2.21
Week 6 51.00 ±3.80 49.00 ±3.80 57.25 ±3.22 42.75 ±3.23
LSD
value
14.465 * 12.055 * 6.294 * 11.685 NS
* (P<0.05), NS: Non-significant.
Table 3. Effect of week in % X and Y after cooling and after freezing
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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CONCLUSION
The study had concluded that it was easy and possible to detect the quantity of sex ratio of each
bull alone by revealing the percentage of SRY and ZFX gene, which had referred to the
percentage of Y-bearing spermatozoa and X-bearing spermatozoa respectively by using real time
PCR. There was significant variation between cooling and freezing mean percentages of semen
in SRY and ZFX gene quantity, which mean the freezing process of semen could cause decrease
in the percentages of SRY and increase in the percentage of ZFX gene (increase probability of
female newborns).
الکشف الکمی للنسبھ الجنسیھ لقصبات السائل المنوی المبرده والمجمده باستخدام تفاعل الدنا المتسلسل
الکمی
باقر جعفر حسن،سھیلھ اونیس حسین ، محمد جبیر
فرع الجراحھ والتولید ،کلیة الطب البیطری، جامعة بغداد ،بغداد،العراق.
الخلاصة
جین لمائة وعشرون ( 120 ) قصبة سائل منوی من ZFX جین و SRY صممت الدراسة لمعرفة التقدیر الکمی لکل من
ثیران نوع ھولشتاین على شکل مجموعتین . تضمنت المجموعة الاولى ( 60 ) قصبة مبردة بدرجة حرارة ( 5) خمسة مئویة،
فی حین کانت المجموعة الثانیة تتضمن ( 60 ) قصبة اخرى اخذت بعد التجمید بالنایتروجین السائل.
DNA جمعت العینات من مرکز التلقیح الاصطناعی / ابوغریب / بغداد. ثم ارسلت الى المختبر لغرض استخلاص ال
( Real time PCR) وتم تصمیم البادئات ثم اجری تفاعل الدنا المتسلسل الکمی (Qiamp (باستخدام عدة استخلاص الدنا
لمعرفة النتائج.
-% بینت النتائج بان ھناک اختلاف ملحوض فی النسبة المئویة الجنسیة للقصبات اثناء التبرید وبعد التجمید (تراوحت مابین 35
للقصبات SRY %53-30 ل ، ZFX 69 ل - للقصبات المبردة و 40 SRY %58-%37 ل ، ZFX %59 بالنسبة ل
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
233
المجمدة غلى التوالی ) . الدراسة تمکنت من الاستنتاج انھ من السھل والممکن الکشف الکمی للنسبة الجنسیة لکل ثور على حدة
کما استنتجت بان عملیة التجمید ممکن ان تؤدی الى تقلیل فی ،(real time PCR) باستخدام تفاعل الدنا المتسلسل الکمی
(% اعلى قیمة 69 ) ZFX اقل قیمة 30 %) وتزید من نسبة جین ) SRY النسبة المئویة لجین
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