Article

1. Al-Anbar J. Vet. Sci., Vol.: 4, Supplement, 2011 ISSN: 1999-6527
Proceedings of First Medical Conference of Medical Colleges (Veterinary Research)
University of Anbar
94
In Vitro Capacitation of Ram Spermatozoa
A. F. Majeed* and M. M. T. Al-Jumaily**
*College of Veterinary Medicine University of Anbar
**College of Agriculture University of Anbar
Summary
Epididymus were collected from Al-Ramadi Slaughter house during the periods
from September 2002 to march 2003. The samples were transported to the Laboratory
with normal saline at a temp of 33-35c within 30 minutes. Semen were collected from
the tail of epididymus by aspiration with 18 gauge needle on 3ml disposable suring
containing 1-2 ml TALP media. The collected media together with spermatozoa were
put in a Petri dish and incubated in Co2 incubator at 35 c, 90% Humidity for 4-6 h.
Spermatozoa evaluated for their maturation (The distal Protoplasmic droplet). Matured
Spermatozoa were washed with buffer Solution. Three times with Centrifugation. Then
0.5 ml of spermatozoa pluse 9.5 ml of TALP media with 10 mg/ml heparin,
Centrifuged, and then 0.5 ml from the bottom spermatozoa incubated in Co2 incubator
at 35c with 45 angle. Head to Head agglutination of Spermatozoa was considered the
Criteriia of sperm Capacitation. The results showed that the procedures give a best
result for sperm maturation and capacitation in vitro. It was concluded that a possibility
of sperm maturation and capacition taken from cauda epididymus of ram from slaughter
house sources.
‫ﻣﺧﺗﺑرﯾﺎ‬ ‫اﻟﻛﺑش‬ ‫ﺣﯾﺎﻣن‬ ‫ﺗﻛﯾﯾف‬
‫ﻣﺟﯾد‬ ‫ﻓرج‬ ‫اﻟﺳﺗﺎر‬ ‫ﻋﺑد‬*‫اﻟﺟﻣﯾﻠﻲ‬ ‫طﻪ‬ ‫ﻣؤﯾد‬ ‫وﻣﺣﻣد‬**
*‫ي‬‫اﻟﺑﯾطر‬ ‫اﻟطب‬ ‫ﻛﻠﯾﺔ‬/‫اﻷﻧﺑﺎر‬ ‫ﺟﺎﻣﻌﺔ‬
**‫اﻋﺔ‬‫ر‬‫اﻟز‬ ‫ﻛﻠﯾﺔ‬/‫اﻷﻧﺑﺎر‬ ‫ﺟﺎﻣﻌﺔ‬
‫اﻟﺧﻼﺻﺔ‬
‫ط‬ ‫ﻋن‬ ‫اﺳﺔ‬‫ر‬‫اﻟد‬ ‫ﯾت‬‫ر‬‫أﺟ‬‫اﻟرﻣﺎدي‬ ‫ة‬‫ر‬‫ﻣﺟز‬ ‫ﻣن‬ ‫ﺑﺦ‬‫ر‬‫اﻟﺑ‬ ‫ﺟﻣﻊ‬ ‫ﯾق‬‫ر‬،‫ـول‬‫ﻠ‬‫أﯾ‬ ‫ﻣن‬ ‫ة‬‫ر‬‫اﻟﻔﺗ‬ ‫ﺧﻼل‬2002‫ـﺎﯾس‬‫ﻣ‬ ‫ـﺔ‬‫ﯾ‬‫ﻟﻐﺎ‬2003.
‫ـ‬‫ـ‬‫ﺳ‬‫ا‬‫و‬‫ﺑ‬ ‫ـﺎت‬‫ـ‬‫ﻧ‬‫اﻟﻌﯾ‬ ‫ـت‬‫ـ‬‫ﻠ‬‫ﻧﻘ‬‫ة‬‫ر‬‫ا‬‫ر‬‫ـ‬‫ـ‬‫ﺣ‬ ‫ـﺔ‬‫ـ‬‫ﺟ‬‫وﺑدر‬ ‫ـﻠﺟﻲ‬‫ـ‬‫ﺳ‬‫اﻟﻔ‬ ‫ـﺢ‬‫ـ‬‫ﻠ‬‫اﻟﻣ‬ ‫ـول‬‫ـ‬‫ﻠ‬‫ﻣﺣ‬ ‫ـﻰ‬‫ـ‬‫ﻠ‬‫ﻋ‬ ‫ـﺔ‬‫ـ‬‫ﯾ‬‫ﺣﺎو‬ ‫ـردة‬‫ـ‬‫ﺑ‬‫ﻣ‬ ‫ـﺔ‬‫ـ‬‫ﯾ‬‫ﺣﺎو‬ ‫طﺔ‬33-35ْ‫ﻰ‬‫ـ‬‫ـ‬‫ﻟ‬‫إ‬‫ـﻲ‬‫ـ‬‫ﻓ‬ ‫ـر‬‫ـ‬‫ﺑ‬‫اﻟﻣﺧﺗ‬
‫ﺧﻼل‬ ‫اﻋﺔ‬‫ر‬‫اﻟز‬ ‫ﻛﻠﯾﺔ‬30‫دﻗﯾ‬‫ﺔ‬‫ﻘ‬.‫ـذﻩ‬‫ﯾ‬‫ﻧﺑ‬ ‫ﻧﺞ‬‫ر‬‫ـ‬‫ﺳ‬‫ﺑ‬ ‫ـﺣب‬‫ﺳ‬‫اﻟ‬ ‫اﺳطﺔ‬‫و‬‫ﺑ‬ ‫ﺑﺦ‬‫ر‬‫اﻟﺑ‬ ‫ذﯾل‬ ‫ﻣن‬ ‫اﻟﻣﻧوي‬ ‫اﻟﺳﺎﺋل‬ ‫ﺳﺣب‬3‫ـﻰ‬‫ﻠ‬‫ﻋ‬ ‫ـﺔ‬‫ﯾ‬‫ﺣﺎو‬ ‫ـل‬‫ﻣ‬
1-2‫ـﻲ‬‫ـ‬‫ـ‬‫ﻋ‬‫اﻟزر‬ ‫ـط‬‫ـ‬‫ـ‬‫ﺳ‬‫اﻟو‬ ‫ـن‬‫ـ‬‫ـ‬‫ﻣ‬ ‫ـل‬‫ـ‬‫ـ‬‫ﻣ‬)TALP(.‫اﻟ‬ ‫ي‬‫ـر‬‫ـ‬‫ـ‬‫ﺗ‬‫ﺑ‬ ‫ـﺎق‬‫ـ‬‫ـ‬‫ﺑ‬‫أط‬ ‫ـﻲ‬‫ـ‬‫ـ‬‫ﻓ‬ ‫ـﻲ‬‫ـ‬‫ـ‬‫ﻋ‬‫اﻟزر‬ ‫ـط‬‫ـ‬‫ـ‬‫ﺳ‬‫اﻟو‬‫و‬ ‫ـﺎﻣن‬‫ـ‬‫ـ‬‫ﯾ‬‫اﻟﺣ‬ ‫ـﻌت‬‫ـ‬‫ـ‬‫ﺿ‬‫و‬‫ـت‬‫ـ‬‫ـ‬‫ﺿ‬‫وﺧ‬ ‫ـﺔ‬‫ـ‬‫ـ‬‫ﻣ‬‫ﻣﻌﻘ‬
‫ـﻧﺔ‬‫ـ‬‫ﺿ‬‫ﺣﺎ‬ ‫ـﺗﺧدام‬‫ـ‬‫ﺳ‬‫ﺑﺎ‬‫ﺑون‬‫ر‬‫ـﺎ‬‫ـ‬‫ﻛ‬‫اﻟ‬ ‫ـﯾد‬‫ـ‬‫ﺳ‬‫اوﻛ‬ ‫ـﺎﻧﻲ‬‫ـ‬‫ﺛ‬5%،‫ـﺔ‬‫ـ‬‫ﺑ‬‫ورطو‬90%‫ة‬‫ر‬‫ا‬‫ر‬‫ـ‬‫ـ‬‫ﺣ‬ ‫ـﺔ‬‫ـ‬‫ﺟ‬‫وﺑدر‬35ْ‫ـدة‬‫ـ‬‫ﻣ‬‫ﻟ‬4-6‫ـﺎﻋﺎت‬‫ـ‬‫ﺳ‬‫ـﺎج‬‫ـ‬‫ﺿ‬‫ﻹﻧ‬
‫اﻟﺣﯾﺎﻣن‬.‫ﺟﺔ‬‫در‬ ‫ﻗﯾﻣت‬ ‫وﻗد‬‫إﻧﺿﺎﺟﻬﺎ‬‫ـﻔﻠﻰ‬‫ﺳ‬‫اﻟ‬ ‫اﻟﻬﯾوﻟﺔ‬ ‫ة‬‫ر‬‫اﻟﻘط‬ ‫ﻣوﻗﻊ‬ ‫ﻋﻠﻰ‬ ‫اﻋﺗﻣﺎدا‬.‫ـﻊ‬‫ﻣ‬ ‫ـﻠت‬‫ﺳ‬‫وﻏ‬ ‫ـﺟﺔ‬‫ﺿ‬‫اﻟﻧﺎ‬ ‫ـﺎﻣن‬‫ﯾ‬‫اﻟﺣ‬ ‫ـذت‬‫ﺧ‬‫أ‬
‫ات‬‫ر‬‫ـ‬‫ـ‬‫ﻣ‬ ‫ـﺛﻼث‬‫ﻟ‬‫و‬ ‫ي‬‫ـدار‬‫ـ‬‫ﻟ‬‫ا‬ ‫ـل‬‫ﺳ‬‫اﻟﻐ‬ ‫ـول‬‫ﻠ‬‫ﻣﺣ‬‫اﻟﻣ‬ ‫ـرد‬‫ط‬‫اﻟ‬ ‫ـﻊ‬‫ـ‬‫ﻣ‬ ‫ـﺔ‬‫ﯾ‬‫ﻣﺗﺗﺎﻟ‬‫ي‬‫ز‬‫ـ‬‫ﻛ‬‫ر‬.‫ـﺎر‬‫ﺑ‬‫اﻻﺧﺗ‬ ‫ـﺔ‬‫ـ‬‫ﺑ‬‫أﻧﺑو‬ ‫ـر‬‫ﻌ‬‫ﻗ‬ ‫ـن‬‫ـ‬‫ﻣ‬ ‫ـﺎﻣن‬‫ﯾ‬‫اﻟﺣ‬ ‫ـذت‬‫ـ‬‫ﺧ‬‫أ‬0.5‫ـل‬‫ـ‬‫ﻣ‬
‫أﺿﯾف‬‫و‬‫إﻟﯾﻬﺎ‬‫ﻋﻲ‬‫اﻟزر‬ ‫اﻟوﺳط‬9.5‫ﻣﻊ‬ ‫ﻣل‬‫إﺿﺎﻓﺔ‬10‫ام‬‫ر‬‫ﻣﺎﯾﻛروﻏ‬/‫ﯾن‬‫ر‬‫ـﺎ‬‫ﺑ‬‫ﻫﯾ‬ ‫ـل‬‫ﻣ‬.‫و‬‫ـن‬‫ﺿ‬‫ﺣ‬‫ـدة‬‫ﻣ‬‫ﻟ‬15‫ـﺔ‬‫ﯾ‬‫او‬‫ز‬‫وﺑ‬ ‫ـﺔ‬‫ﻘ‬‫دﻗﯾ‬45
‫ﺟﺔ‬‫در‬‫ـﻧﺔ‬‫ﺿ‬‫ﺣﺎ‬ ‫ـﻲ‬‫ﻓ‬ ‫ـﺎر‬‫ﺑ‬‫اﻻﺧﺗ‬ ‫أﻧﺑوﺑﺔ‬ ‫ﻓﻲ‬CO25%،90%‫ة‬‫ر‬‫ا‬‫ر‬‫ـ‬‫ﺣ‬ ‫ـﺔ‬‫ﺑ‬‫رطو‬35‫ـﺎر‬‫ﯾ‬‫اﻟﻣﻌ‬ ‫ـﺎﻣن‬‫ﯾ‬‫ﻟﻠﺣ‬ ‫ـﻲ‬‫ﺳ‬‫أ‬‫ر‬‫اﻟ‬ ‫ـﺗﻼزن‬‫ﻟ‬‫ا‬ ‫ـر‬‫ﺑ‬‫أﻋﺗ‬ ‫م‬
‫ﻟﺣدوث‬"‫اﻟﺳﻌ‬‫ﺔ‬"Capacitation‫ـﺎﻣن‬‫ﯾ‬‫ﻟﻠﺣ‬.‫ـﻌﺗﻬﺎ‬‫ﺳ‬‫و‬ ‫ـﺎﻣن‬‫ﯾ‬‫اﻟﺣ‬ ‫ـﺎج‬‫ﺿ‬‫إﻧ‬ ‫ـﻲ‬‫ﻓ‬ ‫ـﺗﺧدﻣﺔ‬‫ﺳ‬‫اﻟﻣ‬ ‫ـﺔ‬‫ﻘ‬‫ﯾ‬‫ر‬‫اﻟط‬ ‫أن‬ ‫ـﺎﺋﺞ‬‫ﺗ‬‫اﻟﻧ‬ ‫ـرت‬‫ﻬ‬‫أظ‬ ‫ـد‬‫ﻗ‬‫و‬
‫اﻟﻣﺧﺗﺑر‬ ‫ﻓﻲ‬ ‫اﻟﺗﺣﺿر‬ ‫ﺳﻬﻠﺔ‬.‫ـﺎب‬‫ﺻ‬‫اﻻﺧ‬ ‫ـﻲ‬‫ﻓ‬ ‫ـﻌﺗﻬﺎ‬‫ﺳ‬‫و‬ ‫ـﺎﺟﻬﺎ‬‫ﺿ‬‫إﻧ‬ ‫ـد‬‫ﻌ‬‫ﺑ‬ ‫ﺑﺦ‬‫ر‬‫ـ‬‫ﺑ‬‫اﻟ‬ ‫ـل‬‫ﯾ‬‫ذ‬ ‫ـﺎﻣن‬‫ﯾ‬‫ﺣ‬ ‫ـﺗﺧدام‬‫ﺳ‬‫أ‬ ‫ـﺎن‬‫ﻛ‬‫ﺑﺎﻻﻣ‬ ‫ـﺗﻧﺗﺞ‬‫ﺳ‬‫أ‬ ‫ـد‬‫ﻗ‬‫و‬
‫ﺟﻲ‬‫اﻟﺧﺎر‬.

2. Al-Anbar J. Vet. Sci., Vol.: 4, Supplement, 2011 ISSN: 1999-6527
Proceedings of First Medical Conference of Medical Colleges (Veterinary Research)
University of Anbar
95
Introduction
One of the important requirements for successful in vitro fertilization was the sperm
capacitation. Capacitation defined as a series of biochemical and biophysical changes
prior to fertilization (1). Capacitation involves the removal of sperm coating material
acquired during epididymal transit or during exposure to seminal plasma and cholesterol
depletion resulting in increased membrane permeability to calcium (2). In vitro washing
of spermatozoa before incubation was reported to accelerate capacitation. Heparin is
used for in vitro capacitation of spermatozoa. It modulates gamete interactions in cattle
and sheep IVE system (3). The aim of this study was designed to show the capcitation
of arm spermatozoa obtained from the tail of epididymis.
Materials and Methods
Epididymus were collected from Al-Ramadi Abattoir during the periods from
September 2002 to march 2003. The samples were transported to lab with normal saline
with temp 33-35c within 30 min, semen were collected from the tail of epididymus by
aspiration with 3 ml disposable suring Containing 1-2 ml of TALP media according to
the size of the tail of epididymus according to Im, etal (4). Then the tail of epididymis
minced with medical scalpel in order to carryout the sperm in the media. The collected
media together with the sperms were pult in a sterile petridish and then transported to be
incubated at 35 c. Samples were taken from the media to observe the degree of
maturation and evaluate the sperm according to the presence of protoplasm droplet and
their position on the sperm. Evaluation of semen included individual motility and sperm
abnormalities. The sperms then were incubated at 5% Co2 ,35 c, 90% relative humidity
for 4-6 hours. In a sterile test tubes with 45 angle without cover.
- In vitro Capacitation of Sperms: Following in vitro maturation and examined
under microscope to see the degree of sperm maturation, Then the sperms leaved in
the media outside the incubator at room temp (23-26c) for 30 minutes. After that the
sperms were washed with buffer solution (at temp 23-25c) [nacl; 9 mg/ ml, BSA; 1
mg/ ml, FCS; 10%, Cryst. Penicillin; 100 I.U./ ML, Streptomycin. Sulphate; 0.1
mg/ml]. 1 ml from semen diluted with washed solution to 10 ml. in a test tubes, then
centrifuged at room temp 1200 R. per. minte/ for 3 minutes for three successive
times. Discard the supernatant fluid and the sperms stays at the bottoms of the test
tubes with a little amounts of media. Addition of 10 ml of TALP media [NACL;
114.0mm, KCL; 3.2mm, Nahco3; 25.0mm, NaH2Po4; 0.3mm, Sodium Lactate;
21.0mm, Cacl2; 2.0mm, Mgcl2; 0.5mm, Sodium Pyruvate; 1.0 mm, Heparin; 10
mg/ml, BSA; mg /ml, Streptomycin Sulphate; 50 mg/ml, Crystalline Penicillin; 100
i.u./ml]. Then Centrifuged again at the same previous manner for one time. 9 ml of
supernatant of the media was removed, Incubated in Co2 incubator for 15 minutes at
35c with 45 angle Head to head agglutination of spermatozoa is considered to be a
Criteria of sperm capacitation(1).
Results and Discussion
The results of this study showed the possibility of sperms maturation taken from the
tail of epididymis in TALP media in vitro. The presence of distal protoplasmic droplet
was the criteria of sperm maturation. Similar observations have been made by Wani (5),
Wani, etal. (6) and Al-Jumaily (7). Several workers indicated that epididymal
spermatozoa have been successfully used from slaughtered rams, many hours after their
death, for IVF of sheep oocytes (1, 5, 7). TALP cultured media for in vitro maturation
of sperms were easily prepared media in the Lab (8). This might be attributed to their

3. Al-Anbar J. Vet. Sci., Vol.: 4, Supplement, 2011 ISSN: 1999-6527
Proceedings of First Medical Conference of Medical Colleges (Veterinary Research)
University of Anbar
96
content of chemical material. The result also showed the criteria of head to head
agglutination, which indicates the sperm capacitation. It has been observed that heparin
stimulates the fertilization rate by improving the efficiency of sperm capacitation in
sheep (9). Heparin apparently binds to sperm and plays a role in the sperm uptake of
calcium (10). Capacitation involves the alternation of the plasma membrane, such as
removal of decapicitation factors, removal of cholesterol, influx of calcium, an increase
in intracellular PH, and increase in protein tyrosine phosphorylation. The Majority of
changes that occur during capacitation involve sperm head; however no morphological
occur. Capacitation is believed to allow the sperm to undergo the a croseme reaction.
The life span of capacitation sperm is limited; as a result, precise timing between
capacitation and the acrosome reaction is necessary for proper fertilization to occur (3).
References
1. Wani, N. A. (2002). In vitro maturation and in vitro fertilization of sheep oocytes.
Review. Small Rum. Res., 44: 89- 95.
2. Dale, B. & Elder, K. (1997). In vitro fertilization. Cambridge University press.
3. Schatten, H. & Constantinescu, G. M. (2007). Comparative reproductive biology.
Black well publishing. PP. 132- 192.
4. Im, K. S.; Kim, H. J.; Chung, K. M. & Park, K. W. (1995). Effects of ovary type,
oocyte grade, hormone, sperm concentration and fertilization medium on in
vitro maturation, fertilization and development of bovine follicular oocytes.
AJAS., 2: 123- 127.
5. Wani, N. A. (1996). In vitro maturation and in vitro fertilization of ovine oocytes.
M.V.SC. Thesis. S.K. University of Agricultural Sciences and Technology.
Sringe ar, Jammu and Kashmir, India.
6. Wani, N. A.; Wani, G. M.; Khan, M. Z. & Salahudin, S. (2000). Effect of oocyte
harvesting technique on in vitro maturation and in vitro fertilization in
sheep. Small Rum. Res., 71- 76.
7. Al-Jumaily, M. M. T. (2003). In vitro fertilization in sheep with certain factors
affecting the technique. M.Sc. Thesis. College Agriculture. Al-Anbar
University.
8. Pavlok, A.; Lucas-Hahn, A. & Niemann, H. (1992). Fertilization and development
competence of oocytes derived from different categories of antral follicles.
Mol. Reprod. Dev., 31: 63- 67.
9. Cox, J. F. & Saravia, F. (1992). Use of a multiple sperm penetration assay in Ovines.
In: Proceeding of the XVII Annual meeting of the Chilean society of animal
production. Chilean, 20- 22 October.
10. Parrish, J. J.; Susko-Parrish, J. L. & First, N. L. (1989). Capacitation of bovine
sperm by heparin: Inhibitory effect of glucose and role of intracellular pH.
Biol. Reprod., 41: 683- 699.