RASPA Vol 2 n 1 Mars 2004 VF

Rev ue Africaine de Santé et de Productions Animales
© 2013 E.I.S.M.V. de Dak ar
•
A rticle originAl
In vitro capacitation of buck spermatozoa for in vitro
embryo production
J. KOUAMO1*, S.D. KHARCHE2, PUJA GOEL2, A.K. GOEL2 et S.K. JINDAL2
1
2
School of Veterinary Medicine and Sciences, The University of Ngaoundere, PO BOX: 454. Ngaoundere-CAMEROON.
C.I.R.G., Makhdoom, PO Farah-281122, Mathura (UP) India.
*
Correspondance et tirés à part , e-mail : [email protected]
Abstract:
The objective of the present study was to evaluate the effect of different capacitation protocols of buck spermatozoa on developmental potency of goat
embryos. A total of 437 COCs were collected by slicing from 112 ovaries. The COCs recovered were visually assessed and graded according to the
cumulus cell investment and morphology of the ooplasm. COCs were matured in TCM-199 supplemented with estradiol 17-Beta (1μg/ml), BSA
embryotested (3mg/mL), 10% fetal bovine serum, 20IU/mL PMSG at 38.5°C and 5% CO2 in an incubator under humidified air for 27h. Fresh semen
samples were collected using the artificial vagina from a fertile pure bred adult Sirohi buck, diluted with 5 ml of TALP medium with 10 µg/ml heparin and
10% FBS, and kept for capacitation in a CO2 incubator at 38.5◦C in three different groups depending of capacitation time: (i) Group I: 20mn (n=109); (ii)
Group II: 45mn (n=97); (iii) Group III: 1hr (n=153). Matured oocytes were processed for in vitro fertilization in mTALP and in vitro culture in CR2 medium in
three groups separately. The maturation rate was 82.15%. The cleavage rates, morula and blastocyst stage in group 1; 2 and 3 were 4.59, 20.00, 20.00;
42.27, 17.07, 17.07 and 54.25, 21.68, 7.22%, respectively. The results indicated that there was a statistical difference regarding cleavage rate between
the three groups. It is suggested that, capacitation of buck spermatozoa in 1 hour are suitable for in vitro embryo production. (RASPA, 11 (1) : 49-52).
Keys – Words: Spermatozoa – Capacitation – Embryos – Goats.
Résumé :
Capacitation des spermatozoïdes caprins pour la production d’embryons in vitro
L'objectif de cette étude était d'évaluer l'effet de différents protocoles de capacitation des spermatozoïdes sur le développement embryonnaire in vitro chez
l’espèce caprine. Un total de 437 COCs (Cumulus Oophorus Complex) a été collecté par ‘slicing’ de 112 ovaires. Les COCs ont été évalués puis classés
selon l'investissement de cellules du cumulus et la morphologie du cytoplasme. Ils ont été mis en maturation in vitro dans le TCM-199 complémenté avec
l'œstradiol 17-Beta (1μg/ml), le BSA embryotested (3mg/ml), 10% de sérum fœtal bovin, 20IU/ml de PMSG à 38,5°C et 5% de CO2 dans un incubateur
sous air humide pour 27h. Le sperme a été recueilli à l'aide du vagin artificiel chez un bouc entrainé et testé fertile de race pure Sirohi, puis dilué avec 5 ml
de milieu TALP supplémenté de 10 µg/ml d'héparine et 10% de sérum fœtal bovin (FBS), puis conservé pour la capacitation dans un incubateur à CO2 à
38,5◦C en trois différents groupes en fonction du temps de capacitation: (i) Groupe I: 20mn (n=109); (ii) Groupe II: 45mn (n=97); (iii) Groupe III : 1hr (n=153).
Les ovocytes matures et les spermatozoïdes capacités ont été fécondés in vitro dans le milieu mTALP et cultivés in vitro en milieu CR2 dans les trois
groupes séparément. Le taux de maturation était 82,15%. Les taux de clivage, morula et blastocyste dans les groupes 1 ; 2 et 3 étaient de 4,59, 20,00,
20,00 ; 42,27, 17,07, 17,07 et 54,25, 21,68, 7,22%, respectivement. Les résultats ont montré que le taux de clivage entre les trois groupes était
significativement différent. La capacitation des spermatozoïdes chez l’espèce caprine en 1 heure est la plus appropriée pour la production d'embryons in
vitro.
Mots-clés : Spermatozoïdes – Capacitation – Embryons – Caprins.
Introduction
In vitro embryo production in goat provides an alternate source
of low cost embryos for transfer in breeding programs such as
MOET. The first kid born after complete IVMFC was reported by
Keskintepe et al. (1994) [20]. The various steps involved in
IVMFC or IVP of goat oocytes are quite similar to those
employed in the cattle or sheep, where IVP is an established
procedure. Three main steps involve in IVP: maturation of the
immature oocytes, fertilization of the matured metaphase II (MII)
oocytes with in vitro capacitated fresh or frozen-thawed semen
and culture of the putative embryos for up to 7-8 days until
formation of blastocysts that can be transferred to recipients or
cryopreserved for future use [5, 6, 11, 13, 25, 42]. The IVP
techniques in goats have been detailed by different groups [1,
5, 10, 23, 30]. Following IVM, in vitro fertilization (IVF) is done
with fresh [14, 40] or frozen-thawed [3, 35] buck semen.
RASPA Vol.11 N01, 2013
Several types of IVF media are used which includes, defined
medium [4, 46], TALP medium [28, 29] and synthetic oviductal
fluid (SOF) medium [37, 39]. Some laboratories use heparin [33,
38], heparin and calcium ionophore [17], caffeine or PHE
(penicillamine, hypotaurine and epinephrine) [15] in capacitation
medium for better fertilization and cleavage. Most IVF studies
use freshly ejaculated spermatozoa and a few IVF trials have
been carried out with frozen-thawed spermatozoa. In vitro
fertilization of the oocytes is usually carried out following 27h
maturation in the IVM medium. Motile sperm are obtained by
centrifugation of frozen-thawed semen in Percoll gradient
(45%/90%) at 500 g for 10 min in room temperature. Percoll
gradient separation of spermatozoa seems to be an effective
means of yielding motile sperm from frozen-thawed semen [35].
Motile sperm are collected from the bottom of the 90% fraction
49
J. KOUAMO et al.
and diluted to a concentration of 107 spermatozoa/ml in
media. Some studies have shown that incubation of
spermatozoa for a further 1h period in media supplemented
with heat-inactivated estrus serum enhances the capacitation
process of spermatozoa (sheep and goat : [5]; deer : [2, 7] ).
Coppola et al. [8] routinely add heat – inactivated sheep serum
(2%) to the IVF medium and incubate oocytes with motile
spermatozoa for 17 hours resulting acceptable levels of
blastocyst development while Freitas et al. [11] obtained motile
spermatozoa by centrifugation of semen on a Percoll gradient
(45%/90%) for 10 minutes at 900g. Viable spermatozoa are
incubated for 30 minutes in medium supplemented with heatinactivated estrus goat serum for their capacitation. This
phenomenon leads to "acrosome reaction" causing the release
of proteolytic enzymes that may assist sperm penetration into
the oocyte. Any agent that causes Ca++ entry into the sperm
acrosome and an increase of pH within the sperm enables
capacitation to be accomplished. The objective of the present
experiment is to determine the optimal time for sperm
capacitation and oocyte fertilization and to observe the effect
on early embryo development of in vitro-matured goat oocytes.
4. In vItro fertIlIzatIon
Materials and Method
The overall average oocytes (A and B) recovered per ovary was
All the chemicals and reagents used were from Sigma Chemical
Company (St Louis, MO, USA) except where otherwise indicated.
Pregnant Mare Serum Gonadotropin (PMSG) Folligon® was from
Intervet, International B. V., Boxmeer-Holland. Fetal Bovine Serum (Cat
N° A15-104) from PAA Laboratories GmbH, The Cell Culture Company
(Austria) and microfilters of 0.22µm (Cat N° SLGV033RS) from MillexGV, Carrigtwohill, Co.Cork, Ireland.
1. HarveSted of oocyteS
Goat ovaries (n=112) were collected from the Agra slaughterhouse just
after the slaughter in 0.9% saline supplemented containing 100IU
penicillin-G and 100µg streptomycin sulphate per ml at 30 to 35°C
within 2-3h of slaughter to avoid any detrimental effect.
Oocytes were harvested by slicing technique [44] and the oocyte
collection medium consisted of Dulbecco’s phosphate-buffered saline
(D-5773) with 3 mg/ml BSA. Recovered oocytes were grade as
excellent (A), good (B), fair (C) and poor (D) quality depending upon the
cumulus investment and cytoplasmic distribution under a stereo zoom
microscope [21]. Only grades A and B oocytes (n=437) were selected
for maturation in this study.
After incubation, 100µl of the sperm pellet was diluted in 750µl FertTALP medium in each group. After 27h of culture, matured oocytes
were separated from the cumulus cells by treating with 0.1%
hyaluronidase enzyme. Drops containing the oocytes were inseminated
with 25–50µl of the final diluted semen in each group so as to obtain a
sperm concentration of 1×106 sperm/ml. The concentration of buck
semen was calculated by haemocytometer. After in vitro insemination,
the oocytes and sperm were co-incubated in each group separately for
18h at 38.5◦C in 5% CO2 incubator in humidified air.
5. In vItro culture
After 18h, presumptive zygotes of third groups were washed in culture
medium CR2 enriched with 10 % FBS to remove sperm cells adhered
to zona pellucida and co-cultured at 38.50C in 5 % CO2 incubator in
humidified air for 9 days.
6. StatIStIcal analySIS
Cleavage rate between the different treatments groups were compared
using the Chi-square test. The level of significance was recorded at the
5% level of confidence [36].
Results
1. HarveSted of oocyteS
3.90±0.57.
2. MaturatIon rate
The morphological maturation rate based on cumulus cell
expansion was 82.15%.
3. cleavage rate and eMBryo develoPMent
Based on capacitation time, cleavage rates (P<0.05), morula
(Picture 1) and blastocyst (Picture 2) production in group 1; 2
and 3 were 4.59, 20.00, 20.00; 42.27, 17.07, 17.07 and 54.25,
21.68, 7.22%, respectively (Table 1).
2. In vItro MaturatIon
Selected Cumulus Oocyte Complexes (COCs) for in vitro maturation
were washed 8-10 times in Oocyte Holding Medium (OHM) containing
TCM-199 with Hepes modification and 3-4 times in maturation medium
consisting of TCM-199 supplemented with estradiol 17-Beta (1μg/ml),
BSA embryotested (3mg/mL), 10% fetal bovine serum, 20IU/mL PMSG
[24]. Finally 10-15 COCs were transferred to a 100-µl drop of
maturation media under mineral oil in a polystyrene culture dish (35mm
x 10mm), previously incubated for 2h in a CO2 incubator. Oocytes were
cultured for 27h at 38.5°C in 5% CO2 incubator in humidified air.
3. SeMen collectIon and SPerM In vItro caPacItatIon
Fresh semen samples were collected using the artificial vagina from a
fertile pure bred adult Sirohi buck. The first and second seminal
ejaculates were examined for volume, colour, consistency and gross
sperm and progressive motility. A sample of 100µl fresh semen was
diluted with 5 ml of sperm-TALP medium with 10 µg/ml heparin and
10% FBS and washed by centrifugation at 1200×g for 5 min. The
supernatant was discarded and the pellet was diluted with 5 ml of
medium and kept for capacitation in a CO2 incubator at 38.5◦C in
three different groups depending of capacitation time: (i) Group I: 20mn
(n=109); (ii) Group II: 45mn (n=97); (iii) 1hr (n=153).
50
Picture 1 : Morula stage (x400)
©Justin
Picture 2 : Blastocyst stage (x400)
©Justin
Discussion
The method of oocyte recovery could affect the efficiency of
IVEP [18]. Different techniques of oocyte collection are
employed in order to obtain maximum oocytes of culturable
quality. Several authors founded that by slicing technique
maximum number of oocytes could be obtained [27, 41, 43,
44]. The overall oocytes A and B obtained is lower than the
latter; resultant debris interferes with the microscopic search
and probably many harvested oocytes were lossed [41, 43].
RASPA Vol.11 N01, 2013
In v itro capacitation of buck spermatozoa for in v itro embry o production
table 1: cleavage and embryo development according to different capacitation time.
capacitation time
total
total
oocyte
cleaved
culturable
2 cells
20mn
109
45mn
97
41(42,27%)b 5(12,19%) 8(19,51%) 8(19,51%) 6(14,63%) 7(17,07%) 7(17,07%)
1h
153
83(54,25%)c 4(4,81%) 25(30,12%) 25(30,12%) 5(6,02%) 18(21,68%) 6(7,22%)
5(4,59%)a 0(0,00%)
Maturation of the oocytes is one of the pre requisites of
successful IVF and for in vitro embryo production. The
morphological features of oocytes are visually assessed during
the selection of immature oocytes for in vitro maturation in
mammals [18]. Maturation rate obtained was similar to those
reported by others studies in goats. So, caprine culture media
supplemented with gonadotropins and estradiol-17ß improved
maturation rates significantly [15, 16, 20, 22, 28, 29, 34, 45].
Based on capacitation time, significantly (P<0.05) higher
penetration and cleavage rate were obtained in 1h of
capacitation as compared to 20 min and 45 min. This is in
agreement with several authors [2, 5, 7]. Presently heparin is
commonly used capacitating agent which is one of the
glycosaminoglycan known to capacitate the bull spermatozoa
in-vitro [26]. Sperm capacitation, fertilization, cleavage and
embryo development rate can be improved by the addition of
heparin in in-vitro fertilization media [9, 15, 19, 29, 31, 35, 38,
46]. Heparin apparently binds to sperm and plays a role in the
sperm uptake of calcium [32]. Some authors incubated
spermatozoa in different times for their capacitation [8, 11].
Further efforts have to be made to standardize the semen
capacitating process to improve the quality and feasibility of invitro produced embryos.
Conclusion
Sperm capacitation is a prerequisite for fertilization, as the
response to an oocyte signal can be initiated only in
capacitated sperm. Therefore, there is further need to
investigate different durations of capacitation as well as
concentrations of capacitating agent for better in-vitro
fertilization and embryo development.
Acknowledgments
First author wish to thank the Department of Science &
Technology (DST) and Ministry of External Affairs (MEA),
Government of India through the Federation of Indian
Chambers of Commerce and Industry (FICCI), for providing
opportunity to conduct this research through the CV Raman
International Fellowship programme for African Researchers in
CIRG, Makhdoom, Farah, Mathura. Authors wish also to thank
Director, Central Institute for Research on Goats, Makhdoom,
Farah, Mathura, for providing necessary facilities and help.
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≤≤≤
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