Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
748
EFFECTS OF FETAL CALF SERUM (FCS) WITH ESTRADIOL
17Β (E2) ON EMBRYO PRODUCTION IN LOCAL
BUFFALOS(Bubalus bubalis)
Ihsan A. H. , Alaa A.Sawad, Husamaldeen A. Al-Salim
College of veterinary Medicine , University of Basrsh , Basrah,Iraq.
key words: Reproduction, Bubalus bubalis, , sperm.
Corresponding Author: ihsan.ali.habeb@gmail.com
ABSTRACT
The study was conducted to investigate the effect of add supplement (FCS and
E2) on culture media (Ham’s F-10 and DMEM) on in vitro maturation , in vitro
fertilization(IVF) and embryo development. This study was conducted at the
laboratories of Theriogenology, Department of Surgery and Obstetrics, College of
Veterinary Medicine, Basrah University, during the period extended from January
2017 to the end of April 2018. The samples of study were female reproductive system
and male testis (150 Ovaries and 30 testes) collected from (Al-Basrah abattoir house)
after slaughter at fifteen minutes. All samples were transported in sterilize and clean
cool boxes at (4-8ºC) within 1-2hrs to the center research unit. Oocytes were collected
by aspiration method. Only grad A and B quality oocytes were selected and incubated
in an appropriate maturation medium (Ham’s F-10 and DMEM) at (38.5 C), 5% CO2
and 95% relative humidity for 24-28 hrs. Spermatozoa were obtained by slicing of
caudal epididymal of buffalo's bull. Sperms with matured oocytes were incubated in
an appropriate maturation medium at (38.5 C), 5% CO2 and 95% relative humidity
for 16 -20 hrs. then the fertilized ova were re-incubated in fresh media with changes
50% of media every day and examined every 24hrs for( 4) days to follow embryonic
development The results showed:
There was high significant (P<0.01) difference in the percentage of Oocytes
maturation in Ham’s F-10 and DMEM with supplement (FCS and E2) media groups
compared with control media groups. The results also showed high
significant(P<0.01) difference in the percentage of Oocytes fertilized in Ham’s F-10
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
749
and DMEM supplement (FCS and E2) media groups compared with control media
groups.
INTRODUCTION
Buffaloes represent an integral part of the agricultural economy in world this
important animal species has provided draft power,milk, meat and hide to millions of
people. However, buffaloes have low reproductive potential which could be related to
the low total number of follicles in the ovary; poor super ovulatory response and high
percentage of atretic follicles (1and 2). Efforts have been initiated in recent years to
augment the reproductive potential of these animals using biotechnology (3). There
are new techniques for improving the reproductive efficiency and enhancing the
production of genetically superior animals. In vitro fertilization (IVF) technology
provides an opportunity to produce embryos for genetic manipulation and embryo
transfer (4). Producing embryos by IVF can be done based on three subsequent
techniques: in vitro maturation (IVM) of Oocytes, IVF of in vitro matured oocytes
and then in vitro culture (IVC) of fertilized oocytes for cleavage up to blastocyst stage
(5and 6). (7) refer that the FCS serves as a protein source and may have hormones
bound to it. (8) who suggest that BSA contains a number of known growth factors
playing an important role in the regulation of oocyte maturation. And also prevents
the hardening of zona pellucida. Moreover, the beneficial action of FCS may be
related to its anti-oxidant properties, which favors the increased maturation rate. (9)
found the percentage of buffalo Oocyte maturation with supplement FCS 67.52
compared with control 40.78 this result refer that supplement of FCS had an important
role on in vitro maturation of buffalo oocytes. (10) was believed that the beneficial
effects of BSA are due to its content of cyclic adenosine monophosphate,
catecolamines, vitamins, putative growth factors, lipids and albumin and it has been
also demonstrated that the beneficial effect of FCS supplementation is due to the
presence of a relatively high molecular weight protein which contributes to
maturation of oocytes.
MATERIALS AND METHODS
Collection of Ovaries. Buffalo ovaries (n=150) were collected from local Basrah
abattoir were collected 15 min after slaughter according to (11) and transported
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
750
immediately to the laboratory in a cold box containing sterile normal saline with
antibiotics (100 IU/mL penicillin, 100 μg/mL streptomycin sulphate) at 4-8°C.
Extraneous tissue was removed to clean the ovaries. Prior to Oocyte collection the
ovaries were rinsed in 70% ethanol to minimize the risk of contamination followed by
three rinses with sterile normal saline to remove the traces of ethanol.
Recovery of oocytes. Follicular Oocytes (n=320) Oocytes were recovered by
Aspiration method:
Oocytes were aspirated from 10-20 mm visible follicles using an 18- gauge needle
attached to a 10-ml disposable syringe containing 1 ml of media. The recovered
compact Oocyte complexes (COCs) were examined and classified according to their
quality (12) into:
Grade 1: Good Oocytes had a homogenous evenly granular Ooplasm and were
surrounded by compact and dense cumulus cell layers.
Grade 2: Fair Oocytes were surrounded by 1-3 layers of cumulus cells.
Grade 3: Denuded Oocytes had uneven Ooplasm and were completely devoid of
cumulus cells around them.
Experiment 1 :-The Oocytes were divided into four groups according to the type
of maturation medium with supplemented:
Group 1: oocytes (n = 80) matured using Ham’s F-10 (control group) (Hycolne,
Logan ,Austria) without supplemented.
Group 2: oocytes (n = 80) matured using Ham’s F-10 Hycolne, Logan Austria)
supplemented with either 10% FCS with 1 mg/ml estradiol 17β (E2).
Group 3: oocytes (n = 80) matured using DMEM (control group) (Sigma)
supplemented without supplemented .
Group 4: oocytes (n = 80) matured using DMEM (Sigma) supplemented with either
10% FCS with 1 mg/ml estradiol 17β (E2).
Only grade 1 and grade 2 were used supplemented with (100 IU/mL penicillin, 100
μg/mL streptomycin sulphate). The culture dishes were placed in a CO2 incubator
(95% relative humidity, 5% CO2 at 38.5ºC) for 24 h. Maturation oocyte was assessed
according to polar body (13) and determined by the degree of cumulus cells
expansion to excellent, described by (14).
Experiment 2:-Spermatozoa maturation and Capacitation:
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
751
After collection of sperm ,the spermatozoa samples transported from petri dish to
10ml test tube containing 2ml maturation media ( TCM-199) with Nystatin and
Penicillin , the test tube was incubated at 37 ºC for 4 hrs , the loose of cytoplasmic
droplet from the tail of spermatozoa was the criteria of sperm maturation (15). To
complete the next step (sperm capacitation) 50 IU/ml heparin were added at the last
45minutes of the time of maturation to complete the step of sperm maturation and
sperm Capacitation (16).
Experiment 3:-In vitro fertilization
Matured Oocytes were washed twice with medium supplied with antibiotics and
antifungal before transferred to a glass Petri-dishes containing IVF medium which is
supplemented with E2, FCS, pencilline- streptomycin and antifungal preparation (17).
Capacitated spermatozoa sample was prepared and diluted to yield 1-2× 106 sperms
needed for fertilization (17). After that, Petri- dishes which containing mixture
Gametes was incubated at (95% relative humidity, 5% CO2 at 38.5ºC) for 14- 18 hrs
(18 and 19). Approximately every 24 h for up to 5 days was checked in order to
confirm the occurrence of fertilization. The cleavage rate was recorded according to
(20) as the total number of cleaved Oocytes/total number of matured cultured Oocytes
multiplied by 100.
Statistical analysis
Chi-square test was used to compare maturation and fertilization rates among
different groups (21). A 1% significance level was used.
RESULTS
1- Effect FCS with Estradiol 17β (E2) on In vitro maturation (IVM) of local iraq
buffalos Oocyte:-
The results indicate that Oocytes maturation percentage increased significantly
value (P< 0.01) in Ham’s F-10 media with 10% FCS and (E2) supplemented
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
752
compared group with Control group 50% (80/40), 13.75% (80/11) respectively. On
the other hand the percentage of oocytes maturation in DMEM with 10% FCS and
(E2)supplemented group increased significantly value (P< 0.01) than the control
group 61.25% ( 80/49) , 16.25% (80/13) respectively . While there was nonsignificantly
value (P> 0.01) between control group and treated group as shown in
table (1 ).
Table (1 ): Effect of FCS with E2 on In vitro maturation (IVM) of local iraq buffalos
oocyte:-
Different small letters vertically denote significant (P<0.01) different between
parameters.
2-Effect FCS with Estradiol 17β (E2) on In vitro fertilization (IVF) of local Iraq
buffalos oocytes:-
The results illustrated that the percentage oocytes fertilization increased
significantly value (P< 0.01) in Ham’s F-10 media with 10% FCS and (E2)
supplemented group compared with Control group 40% (40/16), 18.18% (11/2)
respectively. On the other hand the percentage of embryo development (2, 4, 8 and 16
cell) at 24,48.72 and 96 hrs high significantly value (P< 0.01) in Ham’s F-10 media
Parameter
Media
No-oocytes
culture
No-oocytes
maturated
Percentage
oocytes
maturated
Percentage
oocytes nonmaturated
Ham’s F-10
(Control)
80 11
b
(13.75%)
b
(86.25%)
b
Ham’s F-10
with FCS and
E2
80 40
a
(50%)
a
(50%)
a
DMEM
(Control)
80 13
b
(16.25%)
b
(83.75%)
b
DMEM with
FCS and E
80 49
a
(61.25%)
a
(38.75%)
a
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
753
with 10% FCS and (E2) supplemented group compared with Control group. The
results showed that the percentage oocytes fertilization increased significantly value
(P< 0.01) in DMEM with 10% FCS and (E2) supplemented group compared with
Control group 38.77% (49/19), 23.07% (13/3) respectively. On the other hand the
percentage of embryo development (2 , 4 ,8,16 cell) at 24,48.72 and 96 hrs high
significantly value (P< 0.01) in DMEM with 10% FCS and (E2)supplemented group
compared with Control group . While there was non-significantly value (P> 0.01)
between control group and treated group as shown in (table 2 ).
Table ( 2 ): Effect of FCS with E2 on In vitro Fertilization (IVF) of local iraq buffalos
oocyte:-
Parameter
Media
No-oocytes
maturated
No. and
percentage
zygotes
No. and
percentage
two cells
At 24 hrs.
No. and
percentage
Four cells
At 48 hrs.
No. and
percentage
Eight cells
At 72 hrs.
No. and
percentage
sixteen cells
At 96 hrs.
Ham’s F-
١٠
(Control)
11
b
2
(18.18%)
b
١
(٥٠%)
b
٠
( ٠.٠%)
b
٠
(٠.٠%)
b
٠
(٠.٠%)
b
Ham’s F-
10 with
10% FCS
and (E2)
40
a
16
(40%)
a
9
(56.25%)
a
6
(37.5%)
a
4
(25%)
a
2
(12.5%)
a
DMEM
(Control)
13
b
3
(23.07%)
b
1
(33.33%)
b
0
(0.0%)
b
0
(0.0%)
b
0
(0.0%)
b
DMEM
with 10%
FCS and
(E)
49
a
19
(38.77%)
a
7
(36.84%)
a
5
(26.31%)
a
4
(21.05%)
a
4
(21.05%)
a
Different small letters vertically denote significant (P<0.01) different between
parameters
Fig .1 Two cells Fig .2 four cells
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
754
Fig .3 Eight cells Fig .4 Sixteen cells
DISCUSSION
Many factors affect in vitro maturation of buffalo oocytes. These factors are either
the selection of proper maturation medium, the quality of Oocytes , the hormones
added and serum supplemented (22).
The present study revealed that supplementation of maturation media with FCS with
E2 led to higher maturation and fertilization rates compared with free medium these
results coincide with those of (23) and (18) who observed difference between serum
free group and serum supplemented group for IVM. Moreover, these results are in
agreement with those obtained by (24), (25), (26) and. (27) who recorded beneficial
effect of serum supplementation on in vitro maturation rate of buffalo oocytes. This
might be attributed to the action of serum that promotes cumulus cells-oocyte
uncoupling by retaining the hyaluronic acid within the COCs in a manner that results
in cumulus mucification. This uncoupling could be responsible for stopping the
transfer of oocyte maturation inhibition factor via gap junction (28).
The results of this study revealed that the addition of serum to the TCM-199 medium
enhanced the maturation rate of follicular oocytes. Similar findings have been
reported by (29) in cattle. (30) found a favourable effect of oestrus cow serum when
added to the maturation medium. These authors have postulated that the addition of
sera to the culture medium during the maturation of oocytes promotes the rupture of
germinal vesicle and induces oocyte maturation. Hence, supplementation of the media
with serum had a biphasic favourable effect on maturation. The beneficial effects of
serum for oocyte maturation may also act via cumulus cells or directly on the oocytes.
(31).
Serum may provide beneficial factors to the culture medium, including energy
substrates, amino acids and vitamins. There may be specific effects of serum
component possibly growth factors on oocyte maturation That is manifested as
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
755
improved embryo development following IVF (32). Also, (33) suggested that it may
be important to include serum in the in vitro maturation medium to prevent hardening
of zona pellucida which could adversely affect fertilization. In the same manner, (34)
reported that fetuin, a major glycoprotein constituent of fetal calf serum, can prevent
hardening of zona pellucida during in vitro maturation as it acts by preventing the
action of proteolytic enzymes originating from precociously released cortical granules
and improve the fertilization capacity of oocytes. Also, (35) recorded another
beneficial action of serum which is its antioxidant properties by reducing superoxide
formation. In addition, serum added to the oocyte medium provides a source of
albumin that balances the osmolarity (36). There are different sources for
supplemented sera such as fetal calf serum (37,38), oestrus buffalo serum (39), steer
serum (40) and superovulated buffalo serum (41). Estradiol has been found to
improve the completion of maturational changes and also to support the synthesis of
presumed male pronuclear growth factor (42, 43).
على انتاج (E مع ھرمون الاستردایول ( 2 (FCS) تأثیرات مصل العجول البقری
اجنة الجاموس العراقی المحلی
احسان علی حبیب،علاء عبد الخالق سواد ، حسام الدین السالم
الخلاصھ
المضاف الى (FCS and E ھدفت الدراسة الى تأثیر مصل العجول البقری وھرمون الاستوجین ( 2
على نسبة انضاج واخصاب وانتاج اجنة الجاموس (Ham’s F-10 and DMEM) الاوساط الزرعیھ
المحلی. أجریت الدراسھ فی مختبرات کلیھ الطب البیطری( فرع الجراحھ والتولید وحدة الابحاث المرکزیھ)
جامعھ البصرة للفتره من شھر کانون الثانی لنھایة شھر میسان ٢٠١٨ جمعت نماذج الدراسھ الاجھزه
التناسلیھ الانثویھ للجاموس وخصى ذکور الجاموس (عدد المبایض التی تم الحصول علیھا
١٥٠ مبیض وعدد الخص ٣٠ خصیھ ) بعد الذبح مباشرة بخمسة عشر دقیقة من مجزره البصره
العصریھ. ، حیث نقلت نماذج الدراسھ فی حاویات خاصھ نضیفھ ومعقمھ تحتوی على محلول
٢) ساعة الى وحدة الابحاث - ٨) درجھ مئویھ خلال مدة ( ١ - فسلجی مبرد وبدرجھ حرارة( ٤
أختیرت البیوض .Aspiration المرکزیھ . تم أستحصال البیوض من المبایض بطریقة السحب
فقط لغرض الأنضاج. حضنت البیوض المختارة فی الاوساط الزرعیھ A,B من صنف
فی حاضنھ ثنائی اوکسید الکاربون بنسبھ ٥% والرطوبھ (Ham’s F-10 and DMEM)
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
756
٢٨ ساعة. اما بالنسبھ للحصول على - النسبیة ٩٥ % وبدرجة حراره ٣٨.5 درجھ مئویھ لمدة ٢٤
النطف عن طریق اسئصال ذیل البربخ للثیران. حضنت البیوض والنطف الناضجھ فی الاوساط
% فی حاضنھ ثنائی اوکسید الکاربون بنسبھ ٥ (Ham’s F-10 and DMEM) الزرعیھ
٢٠ ساعة . تم عزل - والرطوبھ النسبیة ٩٥ % وبدرجة حراره ٣٨.5 درجھ مئویھ لمدة ١٦
البیوض المخصبھ واعادتھا الى الوسط الزرعی ، تم متابعھ تطور الاجنھ کل ٢٤ ساعھ مع تغیر
٥٠ % من حجم الوسط الزرعی یومیا بوسط زرعی طازج ولغایة( ٤ ) أیام الى حین تکون
التویثھ ، وقد اظھرت النتائج التالیھ.
Ham’s F-10 and ) فی نسبھ البیوض الناضجھ فی الوسط (P< وجود فرق معنوی ( 0.01
المعامل بمصل العجول البقری وھرمون الاستروجین مقارنة مع نفس الاوساط (DMEM
الزرعیھ الغیر معاملة بمصل العجول البقری وھرمون الاستروجین ، کما تبین من الدراسھ
Ham’s F-10 and ) فی نسبھ البیوض الخصاب فی الوسط (P< وجود فرق معنوی ( 0.01
المعامل بمصل العجول البقری وھرمون الاستروجین مقارنة مع نفس الاوساط (DMEM
الزرعیھ الغیر معاملة بمصل العجول البقری وھرمون الاستروجین
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