summary of the phd thesis researches ...for in vitro fertilization” v for in vitro maturation of...

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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.



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).



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


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.



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).



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.


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



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%).



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”.



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.



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.



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.



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.


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.



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%).



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



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;



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.



XVI

SELECTED REFERNCES

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Chile.

2. CIUPE SIMONA, 2004, Cercetări privind morfologia embrionilor în vederea

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de embriones en oveja. Agro Sur. 12: 6-10;

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Editura “Ion Ionescu de la Brad”, Iaşi;

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treatment regime on ovulatory response in sheep. Proc. Aust. Soc. Reprod. Biol.

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by superovulation treatments. J. Reprod. Fertil. 70, 47-53;

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International: University Press, Cambridge;

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in farm animals series. Vol. 2. CABI Publishing. Cambridge;

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la specia ovină. Ed.Ceres, 1996;

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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;

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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;



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.

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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,

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summary of the phd thesis researches ...for in vitro fertilization” v for in vitro maturation of...

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