summary of the phd thesis researches ...for in vitro fertilization” v for in vitro maturation of...
Post on 06-Oct-2020
2 Views
Preview:
TRANSCRIPT
______________________________________________________________
Cluj-Napoca
2010
UNIVERSITY OF AGRICULTURAL
SCIENCES AND VETERINRY MEDICINE
CLUJ-NAPOCA
DOCTORAL SCHOOL
Faculty of Veterinary Medicine
PETREAN ANAMARIA LUCIANA
SUMMARY OF THE PHD THESIS
Scientific coordinator,
Prof. Univ. Dr. IOAN GROZA
RESEARCHES REGARDING
MORPHOLOGICAL ASSESSMENT OF
SHEEP OOCYTES FOR
IN VITRO FERTILIZATION
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
I
INTRODUCTION
In vitro embryo production of sheep has increased greatly in recent years due to
the expansion and development of new biotechnology applications livestock
(cryopreservation, micromanipulation, embryos sexing, cloning) involved in basic cell
biology research (Davis and Corea, 1984, Celestin, 2003). IVF protocol requires
achievement of the following major steps: oocytes collection from slaughtered or live
animals, in vitro maturation (IVM) in culture media, preparation of semen for in vitro
fertilization (IVF) and in vitro development assurance (IVD ) of zygote obtained
(Lonergan P. et. al., 2000).
PERSONAL RESEARCH
Purpose of the thesis
Given the importance of this particular biotechnology, the research was conducted
on several priorities:
evaluation and identification of the best recovery oocytes techniques from
indigenous sheep breeds;
improvement of morphological and selection methods for sheep oocytes
assessment for in vitro maturation;
assessment degree of maturation after in vitro cultivation by morphological and
morphocitometric aspects, ultrastructural stains;
semen preparation protocol used in the in vitro fertilization of sheep oocytes in
order to sperm capacitation and obtaining a superior fertilization rates;
estimating in vitro fertilization rate of sheep oocytes and studying the
development of the resulting embryos until reaching the early blastocyst stage.
SUPEROVULATION AUTOCHTHONOUS BREEDS OF
SHEEP FOR OOCYTES COLEECTION
PURPOSE OF RESEARCH
Tthe objective of this chapter is the implementation in practice and assessing the
potential use of superovulatory treatments based on PMSG in autochthonous breeds of
sheep breeds of domestic sheep (Transylvanian Merino, Ţigaie, Ţuracnă) in season
and out of season and prices optimization
MATERIAL AND METHOD
The researchs were carried out during April 2007 - July 2010 in the Clinic of
Reproduction, Obstetrics and Gynecology Veterinary of Veterinary Faculty Medicine,
Teaching Experimental Station belonging to the University of Agricultural Sciences and
Veterinary Medicine Cluj-Napoca, and two private farms in the county of Cluj.
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
II
The researches were conducted on a total of 180 Transylvanian Merino sheep,
Ţigaie and Ţurcană, aged between 1,3 and 6 years who subjected to clinical examination
supplemented by paraclinical tests.
To assess the response to hormonal treatments applied were formed the following
experimental batches:
batch I (30 sheep) superovulated in breeding season with intravaginal sponges,
prostaglandin and PMSG administered immediately after sponges extraction;
batch II (30 sheep) superovulated in breeding season with intravaginal sponges
PMSG administered immediately after sponges extraction;
batch III (30 sheep), the control group in breeding season was not superovulated;
batch IV (30 sheep) superovulated in non-breeding season after batch I protocol;
batch V (30 sheep) superovulated in non-breeding season after batch II protocol;
batchVI (30 sheep), the control group in non-breeding season was not
superovulated.
For economic reasons, superovulatory hormonal treatments were repeated three
times successively. Every time was performed the evaluation of follicles number
developed on each ovary.
Assessment of superovulatory treatment response involved median laparatomy
for examination and counting follicles on the ovaries surface.
RESULTS AND DISCUSSIONS
All values obtained by performing metabolic profile falls within physiological
limits, which demonstrates that animals are in healthy condition, both clinically and
laboratory tests.
Applying the two superovulatory methods at three sheep breeds studied, allowed
to obtain superior results in the season (group I, II) to the non- breeding season (group
IV, V), and best results were recorded in Merino sheep, followed by Ţigaie and Ţurcană.
So, in Merino sheep (batch I and IV) it was identified after three harvesting 7,58 vs. 4,85
follicles/ewe, in Ţigaie sheep it was identified 5 vs. 3,46 follicles/ewe and in Ţurcană
sheep it was identified 2,65 vs. 2 follicles/ewe. În Merino sheep (batch II and V) it was
identified 6,07 vs. 4 follicles/ewe, in Ţigaie sheep 4,42 vs. 3 follicles/ewe and in Ţurcană
sheep 2,65 vs. 2 follicles/ewe.
As the two control groups the average number of follicles/sheep/treatment
evaluated is favourable to batch III ((Merinos – 1,92 follicles/ewe, Ţigaie – 1,5
follicles/ewe, Ţurcană - 1,04 follicles/ewe) relatively to batch VI (Merinos – 1,19
follicles/ewe, Ţigaie – 1,04 follicles/ewe, Ţurcană – 0,58 follicles/ewe).
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
III
RECOVERY AND IDENTIFICATION OF SHEEP OOCYTES
PURPOSE OF RESEARCH
The first aspects of in vitro fertilization protocol studied by researchers were the
methods used to obtain and recover oocytes. Given the importance of this stage in the
success of the in vitro fertilization protocol, the purpose of these research was to
appreciate oocytes rate collection according to breed (Transylvanian Merino, Ţigaie
and Ţurcană) and recovery method on superovulated/non-superovulated animals, in
breeding season (September -November) and in non-breeding season (May-August).
MATERIAL AND METHOD
The researches were carried out during April 2007 - July 2010 in the Clinic of
Reproduction, Obstetrics and Veterinary Gynecology, Faculty of Veterinary Medicine
Cluj-Napoca and in a slaughterhouse in the area of Baia Mare, Maramures. Were used to
recover a total of 180 sheep, 30 in each batch. To obtain reliable results, the collections
were repeated three times for each group.
Sheep oocytes recovery from both live and slaughtered was achieved through two
methods: oocytes recovery by follicular aspiration of follicles visible on the surface of
ovary and slicing and trituration of ovaries after bilateral ovariectomy technique.
Sheep oocytes recovery by two collection methods
RESULTS AND DISCUSSIONS
Comparing the results recorded after sheep oocytes recovery from live animals is
observed that those obtained in the breeding season (batch I, II, III) are superior to those
obtained in non-breeding season (batch IV, V, VI) for all breeds of sheep. So, in Merino
sheep (batch I and IV) were aspirated 5,88 vs. 3,3 oocytes/ewe, in Ţigaie sheep were
aspirated 3,75 vs. 2,29 oocytes/ewe and at Ţurcană sheep were aspirated 2,29 vs. 1,71
oocytes/ewe. By slicing and trituration method were recovered 43 oocytes from batch I
and 24 oocytes from batch IV.
In Merino sheep (batch II and V) were collected 4,88 vs. 2,71 oocytes/ewe, in
Ţigaie sheep were viewed 3,38 vs. 1,92 oocytes/ewe and at Ţurcană sheep were
identified 1,88 vs. 1,38 oocytes/ewe. By slicing and trituration method were obtained 32
oocytes from batch II and 19 oocytes from batch V.
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
IV
Regarding the two control groups the mean number of oocytes/ewe identified is
for batch III (Merino – 1,13 oocytes/ewe, Ţigaie – 0,83 oocytes/ewe, Ţurcană – 0,50
oocytes/ewe) versus batch VI (Merino – 0,58 oocytes/ewe, Ţigaie – 0,50 oocytes/ewe,
Ţurcană – o,25 oocytes/ewe). By slicing and trituration method were obtaining 16
oocytes in batch III and 9 oocytes in batch VI.
Comparing the average recorded from oocytes harvested from slaughtered animals
is found that the values obtained in breeding season (batch VII, batch VIII) are superior
to those obtained in non-breeding season (batch IX, batch X) for all breeds of sheep:
in Merino sheep (batch VII and IX) were aspirated 2,15vs.1,55 oocytes/ewe, at
Ţigaie sheep 1,75vs. 1,1oocytes/ewe and at Ţurcană sheep 1,3vs. 0,55 oocytes/ewe;
in batch VIII and X, the average recorded after performing slicing and trituration
method are: in Merino sheep were identified 2,45 vs.1,80 oocytes/ewe, in Ţigaie sheep
1,95vs.1,45 oocytes/ewe and in Ţurcană sheep 1,50 vs. 0,9oocytes / ewe.
MORPHOLOGIC ASSESSMENT OF SHEEP OOCYTES
FOR IN VITRO FERTILIZATION
PURPOSE OF RESEARCH
Oocyte quality has an impact on embryonic development, survival,
transformation, installing and maintaining pregnancy and fetal development. Classical
methods of classification based on morphological classification proved over time
insufficient and subjective because immature oocytes were selected, which led to a small
percentage of viable embryos (Hytell, P. et al., 2000b).
Tthis chapter aims is to improve the morphological assessment system of sheep
oocytes before and after maturation, by introducing the morphocitometric evaluation
and intra-vital and ultrastructural stains in order to select them for vitro fertilization.
MATERIAL AND METHOD
The research was carried out during April 2007 - July 2010 on a total of 1456
oocytes grouped into 10 batches based on race, season and recovery method.
Based on the morphological characters identified (appearance of the zona
pellucida, cumulus ooforus, cytoplasm, perivitelin space) sheep oocytes collected were
classified into two quality categories: „cultivable”oocytes and „non-cultivable” oocytes.
Morphocitometric analysis was performed with the Images Plus Motic microscope
software, studing the zona pellucida and cumulus thickness and diameter oocyte.
Morphocitometric measurements made, allowed us a new reclassification of oocytes
into four quality classes: class I, class II, class III, class IV.
The Brilliant cressyl blue (BCB) test determines the activity of glucose
dehydrogenase - 6 - phosphate (G6PDH), an enzyme synthesized in immature oocytes
but with low activity in mature oocytes. After staining the oocytes were divided into two
categories depending on the degree of cytoplasm staining: oocytes BCB- (colourless
cytoplasm) and oocytes BCB+ (coloured cytoplasm).
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
V
For in vitro maturation of sheep oocytes we used TCM-199 Hepes medium
originally supplemented: 10% FCS, sodium pyruvate, glucose, penicillin, streptomycin,
FSH, LH.
Assessment degree of oocytes maturation was accomplished according to
morphological characters identified. Grown oocytes were grouped into two quality
categories: „mature” oocytes and „degenerate” oocytes.
The morphocitometric measurements (oocyte diameter, expanded cumulus size,
pellucida membrane thickness) gave us the possibility of oocytes reclassification into
four quality classes: „excellent mature” oocytes, „good mature” oocytes, „immature”
oocytes, „degenerate” oocytes.
In order to assess the degree of ultrastructural maturation of sheep oocytes were
performed following stains:
triple staining actin - tubulin - chromatin;
double staining - cortical granules and chromatin;
double staining of mitochondria and chromatin.
RESULTS AND DISCUSSIONS
Comparing the results achieved after morphological assessment of sheep oocytes
recovered from live animals we observed that those obtained in the breeding season
(batch I, II, III) are superior to those obtained in non-breeding season (batch IV, V, VI)
for all breeds of sheep, regardless of collection method applied. Thus, the highest
percentages of „cultivable” oocytes obtained in batch I (83,74%), batch II (80,77%) and
batch III (66,66%). The lowest percentages were in batch VI (55,55%), batch V (73,97%)
and batch IV (77,53%).
„Cultivable” oocytes
The lowest percentage of „non-cultivable”oocytes were recorded in group I
(16,26%), group II (19,23%) and group III (33,33%), while highest percentages were
recorded in group VI (75%), followed by group V (34,21%) and group IV ( 27,66%).
„Non-cultivable” oocytes
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
VI
Regarding the results obtained by morphological assessment of sheep oocytes
from slaughtered animals shows that those obtained in breeding season (batch VII, VIII)
are superior both qualitatively and quantitatively to those obtained in non-breeding
season (batch IX, X). Consequently, in breeding season the highest percentage of
„cultivable” oocytes was 68,48% and the lowest percentage of „non-cultivable” oocytes
was 31,52%. In contrast the percentages of „non-cultivable” oocytes are increased,
43,28%.
We note that morphocitometric examination data obtained from oocytes collected
from live animals are in favor for breeding season (batch I, II, III) than in non- breeding
season (batch IV, V, VI) regardless of collection method used. Regarding individual
values best results were obtained in Merinos, followed by Ţigaie and Ţurcană sheep. The
percentage of oocytes classified as class I and II belonging to batch I (53,85% - 75,95%)
and batch II (55,55% – 73,08%) are higher than those of batch IV (55,32% - 65,17%)
and batch V (44,74%- 61,64%). Lowest rate of oocytes belonging to class I and II
occurred in batch VI (12,5% – 44,44%) and batch III (35,29% - 48,48%).
Sheep oocytes included in class I and II
The lowest rates of oocytes classified in class III and IV were in batch I (24,05%
- 46.15%) and batch II (26,92% - 44,45%) compared with batch IV (34,83% - 44,68%)
and batch V (55,26% - 38,36%). Highest percentages were noted in control groups
(group III - 51,52% - 64,71%, group VI - 55,56% - 87,5%).
Sheep oocytes included in class III and IV
Regarding the results after the morphocitometric exam of oocytes collected from
slaughtered animals we notice that the percentages of oocytes belonging to class I and II
are are for the benefit of batch VII (46,16% - 62,79%) and batch VIII (33,34% -
53,06%) than those in batch IX (33,33% - 41,93%) and batch X ( 21,06%-38,88%).
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
VII
Brilliant cressyl blue (BCB) stain gave us precise information on the viability of
recovered and analyzed morphologically and morphocitometrically oocytes.
Considerable percentage of BCB + oocytes recovered from live animals were
classified in batch I (73,42%) and batch II (67,69%) compared with batch IV (56,18%)
and batch V (54,79%). As control groups, the percentage of BCB + oocytes obtained
from Merino sheep is higher in the group VI (38,88%) compared with group III
(36,36%).
Percentages of BCB- oocytes are also in favor of batches in the breeding season
(batch I, II, III) than in non-breeding season (batch IV, V, VI). Thus, the lowest
percentage of BCB- oocytes were recorded in batch I (26,58%), batch II (32,31%), while
highest percentages were recorded in batch V (73,68 %) and batch IV (57,45%). Merino
sheep belonging to control groups presented a higher rate of BCB- oocytes in batch III
(63,64%) compared with batch VI (61,12%), other values are favorable to batch III.
The results obtained after the BCB staining of sheep oocytes from slaughtered
animals are in favor of batch VII (48,84%) and batch VIII (38,77%) compared with those
in batch IX (32,26%) and batch X (27,78%) .
Stained BCB + and BCB-sheep oocytes
Based on morphological aspects, after cultivation, the oocytes were classified into
two quality categories : 89,96% „mature” oocytes and 10,04% „degenerate”oocytes.
„Mature” oocytes according to morphologic characteristics
Regarding the results obtained after morphocitometric exam 54% oocytes were
classified as “excellent mature”, 29,55% oocytes as “good mature”, 10,19% oocytes as
“immature” class and 6,26% oocytes as “degenerate”.
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
VIII
a. b.
c. d.
Sheep oocytes included in different classes according to morphological aspects: a)
„excellent mature”; b) „good mature”; c) „immature”; d) „degenerated”
Assessment degree of oocytes maturation by ultrastructural stains
1.Triple staining actin - tubulin – chromatin
This stain involved the assessment of normal/abnormal configuration of actin
band, microfilaments, projections, meiotic spindle microtubules and chromosome
organization metaphases plate.
a b c
Various configurations of abnormal actin microfilaments, 60x.
a) normal configuration; b), c) points of rupture (arrow) heterogeneous distribution
of actin filaments in the cytoplasm
Normal configuration of meiotic spindle microtubules
Meiotic spindle was considered normal only when it was located on the periphery
of oocytes and oriented radially (characteristic appearance of "barrel", with slightly
pointed poles). Abnormal meiotic spindle was classified according to the presence of
severe destructions of microtubules/reduction or absence of fluorescein.
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
IX
Meiotic spindle well structured, 60x Meiotic spindle disorganized, 60x
Normal chromosome metaphases plate organization
Chromosomal organization is considered normal when they form an orderly and
compact plate lined in the spindle equatorial plane. Dispersed or diffuse chromosomes
that formed a discrete group, less defined, were treated as abnormal.
Normal organization of chromosomes
in the equatorial plate, 60x Absence of meiotic spindle, 60x.
2.Double staining - cortical granules and chromatin
Status assessment of cortical granules (CG) and the chromatin was achieved by
two fluorescent probes, cachuete lectin (PNA)FICT conjugated (L-7381) and propidium
iodide (PI). This staining allowed oocytes classification into four grades after distribution
made by Izadyar F. et al., 2008: category 1, category 2, category 3, category 4.
a b c d
Classification of sheep oocytes by the distribution of cortical granules and chromatin: a)
category 1, b) category 2, c) category 3, d) category 4.
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
X
3.Double staining of mitochondria and chromatin
Ultrastructural confirmation of changes occurring during cultivation of oocytes
was performed using CMTM Ros MitoTracker Orange (M-7510, probes, Eugene)
reagent. After confocal examination oocytes were distributed in four grades: category 1,
category 2, category 3 category 4.
a b c d
Classification of sheep oocytes by ultrastructural changes in mitochondria and chromatin:
a) category 1, b) category 2, c) category 3, d) category 4.
INFLUENCE OF MORPHOLOGICAL ASSESSMENT OF OOCYTES
ON IN VITRO FERTILIZATION PROTOCOL
PURPOSE OF RESEARCH
Understanding the basic mechanisms of fertilization was achieved in 1951 by
Austin and Chang when they descovered semen capacitation. Origins of in vitro
fertilization are found since 1959, when Chang showed that in vitro fertilized rabbit
oocytes can develop normal product (Chang M.C., 1968, Chang M.C. et al., 1977). The
embryo is a spherical-shaped structure composed of blastomere cells surrounded by a
jelly-like membrane which constitutes the acellular matrix and which represents the
pellucid zone (Simona Ciupe, 2004).
Consequently, purpose of this study is to study the influence of morphological
assessment of oocytes on in vitro fertilization protocol. By default, the research focused
on improving the preparation techniques of semen, oocytes used in IVF protocol and
evaluation of original supplemented culture media used in embryo cultivation.
MATERIAL AND METHOD
The research was carried out during April 2007 - July 2010 in the Biotechnology
Laboratory of the Department of Reproduction, Obstetrics and Veterinary Gynecology of
Faculty of Veterinary Medicine, Cluj - Napoca. In vitro fertilization protocol was
performed on 558 mature oocytes obtained after in vitro cultivation.
Semen collection was performed using Electroejaculation method. Macroscopic
examination (volume, color, smell), was performed immediately after collection and was
supplemented by a microscopic examination which covered the following issues:
viability, morphology, mobility and sperm concentration.
Extender used for dilution and preservation of semen was TRILADYL. Extender
was prepared in accordance with instructions issued by the producing company,
Minitübe, Abfüll-und Labortechnick GmbH&Co. KG, Tiefenbach, Germany.
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
XI
Sperm selection (capacitation) was done through the Swim-up method described
by Solvas I. et al., 2002 using a standardized medium - Sperm TALP. Of deposit
obtained after centrifugation was determined by laboratory tests mobility and viability of
semen and sperm concentration/ml needed for in vitro fertilization of sheep oocytes.
Starting from the composition of an known medium for in vitro fertilization of
sheep oocytes – TALP medium (Tyrode's Albumin Lactate Pyruvate medium) we have
prepared in the laboratory three other media (FI, FII, FIII) original supplemented.
In the protocol of in vitro fertilozation we used two distinct categories of fertilized
sheep oocytes obtained after in vitro cultivation:
category I represented the oocytes classified as „mature” based on
morphological examination;
category II represented the oocytes classified as „mature” based on
morphological examination completed with morphocytometric and ultrastructural
examination;
In vitro cultivation of fertilized sheep oocytes was done in two culture media
(CI and CII) originally supplemented and enriched with various substances. The plates
with presumed zygote were placed in an incubator for 24 hours at 39 0C, 5% CO2, 5%
O2, 90% humidity.
After 24, 48 and 72 hours of incubation, culture plates were examined at inverted
microscope. To determine the stage of embryo development after culture were studied
these issues (Ciupe Simona, 2004): embryo size, appearance and number of blastomere,
compaction degree of the internal cell mass, occupancy of perivitelin space.
RESULTS AND DISCUSSIONS
To achieve in vitro fertilization protocol we selected a total of six rams, bred
Ţurcană, aged 4-6 years. Electroejaculation method allowed obtaining semen from four
rams. Laboratory tests conducted from refrigerated and diluted semen allowed us to
conclude that semen parameters used for in vitro fertilization of sheep oocytes were fell
within the normal range of species.
In the protocol we used two distinct categories of fertilized sheep oocytes obtained
after in vitro cultivation:
category I that includes 279 oocytes classified as „mature” based on
morphological examination; category II that includes 279 oocytes classified as „mature” based on
morphological examination completed with morphocytometric and ultrastructural
examination; Comparing the results of in vitro fertilization of sheep oocytes from category I,
we remarked those obtained in FI medium where fertilization rate was 65,60%, followed
by FIII medium and FII supplemented, where fertilization rates were 59,14% and
46,24%. The lowest percentage of unfertilized oocytes was recorded in FI medium,
15,05% while in FII and FIII were the highest rate of unfertilized oocytes, 21,51%
respectively 18,28%. Degenerated rates of oocytes after fertilization have ranged
between 19,35% in FI medium and 32,25% in FII medium.
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
XII
Fertilized oocytes,
category I
Unfertilized oocytes,
category I
Degenerated oocytes,
category I
Comparing the results of in vitro fertilization of sheep oocytes from category II,
we observed those obtained in FI medium, where fertilization rate was 74,20%, followed
by FIII and FII medium, where fertilization rates were 66,66% and 54,84%. The lowest
percentage of unfertilized oocytes was recorded in FI medium, 10,75%, while in FII and
FIII medium were recorded the highest of unfertilized oocytes, 18,28% respectively
13,98%. Degenerated rates of oocytes after fertilization have ranged between 15,05% in
FI medium and 26,88% in FII medium.
Fertilized oocytes,
category II
Unfertilized oocytes,
category II
Degenerated oocytes,
category II
Cumulus cell removal was achieved by vortexing and fertilized oocytes subjected
to vortex were 100% denudated.
After cultivation sheep embryos were morphologically examined in order to
classify them based on structural aspects into two categories of quality: intact embryos
and degenerated embryos.
Results obtained after in vitro fertilization of mature sheep oocytes from category
I and embryos classification are:
in CI medium, in which 43 fertilized oocytes were cultured, 29 intact embryos
(72,50%) were obtained and 13 degenerated embryos (27,50%);
in CII medium, in which 43 fertilized oocytes were cultured, 25 intact embryos
(62,50%) and 15 degenerated embryos (37,50%).
Results obtained after in vitro fertilization of mature sheep oocytes from category
II and embryos classification are:
in CI medium, in which 43 fertilized oocytes were cultured, 32 intact embryos
(80%) were obtained and 8 degenerated embryos (20%);
in CII medium, in which 43 fertilized oocytes were cultured, 28 intact embryos
(70%) and 12 degenerated embryos (30%).
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
XIII
Stage of embryos development after cultivation was determined according to:
embryo size, appearance and number of blastomere, compaction of internal mass, degree
of occupation of the perivitelin space, the presence of the blastocelic cavity.Thus, from
29 intact embryos cultivated in CI medium, 10 embryos developed to 2-8 cell stage, 15
embryos to morula stage and the remaining 4 embryos to the young blastocyst.
Regarding the stage of development of embryos cultured in CII medium is noted that of a
total of 25 intact embryos, 12 embryos developed to 2-8 cell stage, 12 embryos to morula
stage and 1 embryo in the young blastocyst stage.
The results obtained in category II are: from the 32 intact embryos identified in CI
medium, one embryo developed to 2-8 cell stage, 6 embryos to morula stage and 25
embryos to the young blastocyst stage. The results obtained in CII medium are: 3
embryos developed to 2-8 cell stage, 8 embryos to morula stage and 17 embryos to
young blastocyst stage.
FINAL CONCLUSIONS AND PRACTICAL RECOMANDATIONS 1. After applying the two superovulation protocols we observed that the vaginal
sponges treatment, prostaglandin and equine serum gonadotrophin applied to batch I (413
follicles) and batch IV (277 follicles) allowed obtaining a higher superovulatory response
to vaginal sponges and prostaglandin method applied to batch II (342 follicles) and batch
V (234 follicles).
2. Regarding the applying moment of superovulation in all batches, best response
was obtained during September to November (breeding season) compared with the
period from May to August (non-breeding season).
3. The results obtained in control groups, group III (63 follicles) and group VI (36
follicles) have been decreasing over the lots on which we applied saperovulation
methods.
4. Superovulatory treatment performed in the three breeds of sheep in the breeding
season by the two methods allowed to obtain a higher number of oocytes (batch I - 329
oocytes, batch II - 275 oocytes) compared with groups subjected to the same therapeutic
protocol, in non-breeding season (batch IV - 199 oocytes, batch V - 163 oocytes). Ratio
is the same for control groups.
5. The number of oocytes recovered from slaughtered animals in breeding season
(batch VII - 104 oocytes, batch VIII - 118 oocytes) is higher than in non-breeding season
(batch IX- 68 oocytes, batch X - 84 oocytes).
6. In breeding season the proportion of „cultivable” oocytes was higher (77,76%)
compared with the non-breeding season (70,22%), for all breeds of sheep, regardless
method of collection and treatment applied. Percentage of „non-cultivable”oocytes is for
oocytes recovered breeding season (22,24%) compared with non-breeding season
(29,76%).
7. The morphocytometric results obtained in breeding season from both live and
slaughtered animals were higher (355 oocytes in class I, 195 oocytes in class II, 155
oocytes in class III of, 196 oocytes in class IV) that those recorded in non- breeding
season (156 oocytes in class I, 115 oocytes in class II, 114 oocytes in class III, 164
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
XIV
oocytes in class IV), for all breeds of sheep studied, regardless method of collection and
treatment applied.
8. Values recorded after application of Brilliant cressyl blue stain to oocytes
recovered by both live and slaughtered animals are in favor of breeding season (471
oocytes classified BCB + and 430 oocytes BCB-) compared with those in non-breeding
season (216 oocytes classified BCB + and 339 oocytes BCB-), for all breeds of sheep
included in the study, regardless the method of collection and treatment applied.
9. Morphological characters determined after cultivation by stereomicroscope and
inverted microscope allowed classification of oocytes into two quality classes: 618
„mature”oocytes (89,96%) and 69 „degenerate” oocytes (10,04%). 10. Morphocytometric measurements performed after cultivation, associated with
morphological examination, allowed us reclassification quality oocytes into four classes:
371 “mature excellent” oocytes (54%), 203 “good mature” oocytes (29,55%), 70
“immature” oocytes (10,19%) and 43 “degenerate” oocytes (6,26%).
11. Normal/abnormal evaluation of actin band, meiotic spindle microtubules
configuration and normal/abnormal distribution of actin microfilaments and organization
of metaphase plate chromosome was achieved through actin-tubulin- chromatin stain.
12. Double staining, cortical granules and chromatin, allowed oocytes classification
into four categories: category 1, category 2, category 3, category 4.
13. Mitochondrial and chromatin ultrastrustural changes occurring during oocytes
cultivation allowed the distribution of oocytes in four categories: category 1, category 2,
category 3, category 4. 14. Comparing the results of in vitro fertilization of mature oocytes from category I,
we observed that those obtained in FI medium where higher (65,60%) that those obtained
in FIII medium (59,14%) and FII (46,24%).
15. Analyzing the values derived from in vitro fertilization of sheep mature oocytes,
category II, we observed that those obtained in FI medium, 74,20% (69 oocytes), were
superior to those obtained in FIII and FII medium, where fertilization rates were 66,66%
(62 oocytes) and 54,84% (51 oocytes);
16. Morphological assessment of embryos derived from oocytes belonging to
category I, in the two culture mdium, show structural integrity percentage between
62,50% (25 embryos) and 72,50% (29 embryos) and embryonic degeneration percent
between 27,50% (13 embryos) and 37,50% (15 embryos);
17. After assessment stage of embryos derived from oocytes in category I, at 24
hours were intentified an average of 11 embryos in stage 2, 4 and 8 cells at 48 hours an
average of 13,5 early morula stage and at 72 hours an average of 2,5 embryos in the early
blastocyst stage;
18. Morphological assessment of embryos derived from oocytes belonging to
category II, showed satisfactory percentage ranging from 70% (28 embryos) to 80% (32
embryos) and low percentage of embryonic degeneration, between 20% (8 embryos) and
30% (12 embryos);
19. After the assessment stage of embryos derived from oocytes in category II, at 24
hours was identified an average of 2 embryos in stage 2, 4 and 8-cell, at 48 hours an
average of 7early morula stage and at 72 hours an average of 21 early blastocyst stage;
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
XV
PRACTICAL RECOMMENDATIONS 1. We recommend the use of superovulatory method based on a hormonal
combination of Chrono-Gest + prostaglandin + PMSG and regarding the time of
application we recommend the breeding season.
2. Regarding the breed of sheep that are most suitable for oocytes collection we
recommend the Transylvanian Merino, knowing that improved breeds respond better to
hormonal therapy.
3. Our researchs on the sheep oocytes collection by the two methods, follicular
aspiration and ovary slicing and trituration, allow us to recommend the first method:
superior quality of collected oocytes, even if quantitatively their number
was slightly lower (without statistical significance);
possibility of repeated oocytes recovery from animals without any negative
effects on fertility of donors.
4. For slaughtered animals the success depends on season, the proposed new version
allows harvesting oocytes throughout the year.
5. We recommend improving viability appreciation system of sheep oocytes before
maturation by introducing in the practice intravital Briiliant cressyl blue stain due to the
ease of this technique, low cost and accurate price data. Also we recommend applying
ultrastructural staining after in vitro cultivation of sheep oocytes (triple stain actin-
tubulin-chromatin, double staining, cortical granules and chromatin and mitochondria
and chromatin staining). This stains reflect mature oocytes structures characteristic:
actin band and meiotic spindle microtubules, actin microfilaments distribution and
organization of metaphases plate chromosomes, cortical granules and chromatin and
changes occurring at mitochondrial level.
6. We recommend the use for various biotechnologies (IVF, IVC, cryopreservation)
only of mature sheep oocytes with the following morphocitometric characteristics:
minimum oocyte diameter 110 µm, compaction and cumulus expansion in size over 40
µm, intact pellucida membrane, thick least 13 mm, homogeneous cytoplasm, agranular,
uniform perivitelin space.
7. We recommend for embryos cultivation the use of SOF medium with BSA, lactic
acid, sodium pyruvate and glutamine, to ensure proper embryonic development.
8. For a better efficiency of in vitro fertilization protocol and in order to achieve
superior results, we recommend the use for this technique only of mature oocytes whose
maturation grade was based on morphological examination in conjunction with
morphocytometric and ultrastructural examination.
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
XVI
SELECTED REFERNCES
1. CELESTINOS, M., 2003, Evaluación de la sobrevivencia in vitro de embriones de
coneja bipartidos antes y después de la vitrificación. Tesis de Magister en
Ciencias Mención Reproducción Animal. Universidad Austral de Chile. Valdivia,
Chile.
2. CIUPE SIMONA, 2004, Cercetări privind morfologia embrionilor în vederea
conservării şi transferului embrionar, Teza de Doctorat
3. DAVIS, I.C., J.E. CORREA, 1984, Inducción de superovulación y transferencia
de embriones en oveja. Agro Sur. 12: 6-10;
4. DRUGOCIU, D.G., L.G. RUNCEANU, 2004, Optimizarea reproducţiei la ovine,
Editura “Ion Ionescu de la Brad”, Iaşi;
5. EPPLESTON, J., R.J. BILTON, N.W. MOORE, 1984, Effects of FSH dose and
treatment regime on ovulatory response in sheep. Proc. Aust. Soc. Reprod. Biol.
16, 68;
6. EVANS, G., D.T. ARMSTRONG, 1984, Reduction of sperm transport in ewes
by superovulation treatments. J. Reprod. Fertil. 70, 47-53;
7. GORDON, I., 1994, Laboratory production of cattle embryos, CAB
International: University Press, Cambridge;
8. GORDON, I., 1997, Reproduction in Sheep and Goats. Controlled reproduction
in farm animals series. Vol. 2. CABI Publishing. Cambridge;
9. GROZA, I., Actualităţi şi perspective în biotehnologia transferului de embrioni
la specia ovină. Ed.Ceres, 1996;
10. GROZA, I., M. CENARIU, R. POP, EMOKE PALL, ANAMARIA PETREAN,
2009, Cercetări privind morfometria ovocitelor de scroafă în vederea fecundaţiei
in vitro, Buletinul Societăţii Române de Biologie Celulară, nr. 37, Sesiunea
Ştiinţifică anuală a Societăţii Române de Biologie Celulară, Bistriţa;
11. HYTTEL, P., K.P. XU, T. GREVE, 2000b, Ultrastructural abnormalities of in
vitro fertilization of in vivo matured bovine oocytes, Anat Embryol, 178: 47 – 52;
12. HYTTEL, P., T. GREVE, H. CALLESEN, 2000a, Ultrastructure of in vivo
fertilization in superovulated cattle, J Reprod Fert, 82: 1 – 13;
13. IZADYAR, F., B. COLENBRANDER, M.M. BEVERS, 1997, Stimulatory
effect of growth hormone on in vitro maturation of bovine oocytes is exerted
through the cyclic adenosine 3'-5'-monophosphate signaling pathway, Biology
Reproduction. 57:1484-1489;
„Researches regarding morphological assessment of sheep oocytes
for in vitro fertilization”
XVII
14. IZADYAR, F., W.J. HAGE, B. COLENBRANDER, M.M. BEVERS, 2008, The
promontory effect of growth hormone on the developmental competence of in vito
matured bovine oocytes is due to improved cytoplasmatic maturation. Molecular
reproduction and Development, 49(4): 444-453;
15. MOORE, J.W., J.N. SHELTON, 1964, Egg transfer in sheep, effect of degree of
synchronization between donor and recipients, age of egg, and site of transfer on
the survival of transferred eggs. J. Reprod. Fertil. 7, 145-152;
16. ROBINSON, T. J., 1951, The control of fertility in sheep. II. The augmentation
of fertility by gonadotrophin treatment of the ewe in the normal breeding season.
J. Agric. Sci. 41: 6 – 63;
17. SOLVAS, I., M. GROSSMANN, J. SANTALÓ, M. PONS, 2002, Estudio
comparativo entre dos métodos de Swim-up. Asebir 7: 28-32;
18. WRIGHT, R.W.JR., K. BONDIOLI, J. GRAMMER, F. KUZAN, A. Jr.
MENINO, 1981, FSH or FSH plus LH superovulation in ewes following estrus
synchronization with medroxyprogesterone acetate pessaries. J. Anim. Sci. 52,
115-118;
top related