Effects of needle tip bevel and aspiration procedure on the morphology and developmental capacity of bovine compact cumulus oocyte complexes

ELSEVIER

EFFECTS OF NEEDLE TIP BEVEL AND ASPIRATION PROCEDURE ON THE MORPHOLOGY AND DEVELOPMENTAL CAPACITY OF BOVINE COMPACT CUMULUS OOCYTE COMPLEXES P.E.J. Bolsl

M.T. Ysebaeti:

1

ZDepartment Department

Received

A. Van Soom’

and A. de Kruif’

of Obstetrics, Reproduction and Herd Health of Physiology, Biochemistry and Biometrics Faculty of Veterinary Medicine University of Ghent, Belgium for publication: Accepted:

June

28,

1996

September

26,

1996

ABSTRACT Effects of the needle tip bevel and the aspiration procedure on the morphology of cumulusoocyte-complexes (COCs) and the developmental capacity of the oocytes after IVF were studied in 2 in vitro oocyte pick-up (OPU) simulations using a disposable ovum pick-up needle guidance system. In Experiment I, the influence of the length of the needle bevel was investigated using a short and a long bevelled 20-g disposable needle. After being aspirated from slaughterhouse ovaries, the retrieved COCs were divided into 3 categories: 1) oocytes surrounded by a compact cumulus, 2) oocytes with an expanded cumulus, 3) partially naked oocytes. In Experiment 2, the influence of 5 different levels of aspiration vacuum for 3 different needle diameters (18-g, 19-g, 20-g) and 2 different needle bevels (long, short) was tested on the recovery and on the morphology of the cumulus investment of a fixed number of previously scored compact cumulus oocytes complexes (CCOCs), retrieved after slicing slaughterhouse ovaries. The re-retrieved COCs were allocated to Categories 1 and 3. The results show that the length of the needle bevel has a significant effect on oocyte recovery, in favor of the long-bevelled needle. As soon as higher aspiration vacua are used, a decrease of the number of CCOCs can be observed, which is less prominent for the short-bevelled needle compared to the long-bevelled one. The final number of blastocysts is similar for both needle types. In Experiment 2, the disposable needle system proved to be highly effective since nearly 80% of the CCOCs were retrieved. At low aspiration vacuum, up to 90% of the CCOCs withstand the aspiration procedure undamaged. Increasing the aspiration vacuum results in a decrease of the number of CCOCs, which is less pronounced using thinner needles. Averaged over all needle types, the prevalence of blastocysts expressed relative to the number of recovered oocytes decreases with higher aspiration vacuum. 0 1997 by ElsewerScience Inc Key words: oocyte aspiration, compact cumulus oocyte complex, disposable needles, assisted bovine reproduction

Acknowledgments The authors thank G. Vanroose (IWT), A. Vanhoucke-De Medts and A. Van de Velde (IWONL) for their assistance with IVF procedures; J.M.M. Vandenheede for the excellent technical assistance; and T Van Laere for taking the pictures.

Theriogenology 47:1221-1236. 1997 0 1997 by Else&r Scmce Inc.

0093-691X/97/$17.00 PII SOO93-691X(97)00102-7

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Theriogenology

INTRODUCTION Transvaginal oocyte pick-up (OPU) is gaining popularity worldwide as an important technique for oocyte retrieval in living, previously selected, often genetically highly valuable donor cows. Its high repeatability turns it into a routine procedure in combination with in vitro fertilization (IVF) in assisted bovine reproduction. Once a well running IVF system is established, OPU is believed to become a valuable alternative for classical embryo transfer (ET) programs (10). Up till now it has been used both in the treatment of infertility patients (3, 9, 11) and in young healthy animals as a modem reproduction tool to shorten generation intervals and to hasten the spread of excellent characteristics in a herd (19). The final aim of the transvaginal oocyte pick-up technique is to retrieve as many good quality oocytes as possible. Practical approaches to optimize the recovery of oocytes can be divided into 2 main categories: 1) the biologically-mediated modifications in ovarian dynamics and 2) technical improvements in OPU devices (2, 17, l&21,22). Within the first group, there are different ways to increase oocyte recovery rates such as hormonal stimulation prior to OPU (6, 12, 14, 16, 20), performing OPU at a specific time during the estrous cycle (6, 15, 24) or increasing the number of sessions within a certain time period (8, 25). Although evaluating these biological influences will certainly be a part of the solution to more and good quality oocytes, the more technical aspects cannot be overlooked. No matter how many oocytes can be recruited in vivo, there is a need for a reliable, economically justified oocyte retrieval system. Several research groups refined the transvaginal OPU devices over the last years from a practical point of view, but the actual influence of the aspiration vacuum and needle diameter on the morphology of the cumulusoocyte-complexes (COCs) and on the subsequent developmental capacity of the oocytes has not been thoroughly evaluated. Previous experiments (4) showed that when using disposable needles the aspiration pressure and needle diameter could have a profound influence on the integrity of the cumulus cells surrounding the oocyte, with high aspiration pressures resulting in a higher proportion of naked oocytes. Thinner needles used at moderate aspiration pressures gave the highest proportion of compact cumulus oocyte complexes (CCOCs). Apart from the needle length and diameter, the length of the needle tip or bevel can be altered. The first experiment of this study investigates the possible influence of the needle bevel on the oocyte recovery and on the cumulus oocyte complex morphology. As stated before (4), the oocyte can theoretically appear in different states in the surrounding follicle. It can be surrounded by a compact cumulus or an expanded cumulus, or it may possibly even be naked already. Furthermore, the use of hormonal stimulation can alter the morphology of the COC since this stimulation can cause asynchrony between the maturation of the oocyte and the surrounding follicle (7), or even within the oocyte between nuclear and cytoplasmic maturation (5). Therefore, it would be useful to investigate the net effect of the aspiration procedure on the oocytes surrounded by a compact investment of cumulus cells. This would give us better understanding of the damage the procedure causes, and identify the proportion of oocyte complexes which are already in an inferior state prior to OPU, having

Theriogenology

1223

expanded cumulus cells or being naked from the start. Thus, the aim of the second experiment was to investigate the net effect of the OPU procedure on the morphology of COCs by aspirating oocytes which had been previously

retrieved

from slaughterhouse

ovaries after slicing and scored

as ccocs. MATERIALS Experimental Experiment

Figure

Design 1

1.

Different

In Experiment complex

AND METHODS

morphology

needle types used in Experiment

1, the influence and subsequent

Thus an OPU simulation

1 and 2

of the needle tip bevel on the recovery developmental

was set up using a disposable

capacity

rate, cumulus oocyte

of the oocyte

needle guidance

was investigated.

system which allows for

the use of the needle and tubing system separately, so that the aspiration conditions are the same both in vitro and in vivo (4). Two types of commercially available disposable needles (Terumo Neolus, Leuven, Belgium) were used: 1) the short bevelled 20-g2”3/16 with a length of 55 mm and an external diameter of 0.9 mm (2OS), and 2) the long bevelled 20-g2” with a length of 50 mm and an external diameter of 0.9 mm (20L; Figure 1). The 10% difference in needle length is caused by reasons

of commercial

availability

of the needles.

A stainless

steel connector

was

1224

Theriogenology

used to connect the needles by means of silicone tubings (Silclear TM Tubing, Multi Purpose Medical Grade Silicone Tubing, Degania Silicone/ Degania Bet, Israel) to a plastic 50-ml collection tube (Falcon 2074) and a suction pump with variable suction power (Labotect, Bovenden-GWingen, Germany). The used aspiration vacua were 50, 70, 90, 110 up to 130 mm Hg for each of the 2 needles. For purposes of repeatability of trials and to obtain a sufficient number of follicles to puncture under the same conditions, 106 slaughterhouse ovaries were used. The method of collecting the ovaries was described before (23). A total of 2000 follicles was aspirated, 100 visible follicles (between 3 and 8 mm in diameter) were punctured with the 2 different types of needles at 5 different levels of aspiration vacuum, for a total of 10 different treatment groups (needle bevel by level of aspiration vacuum). Aspiration of the follicles happened as described before (4). To test the repeatability of the experimental set up, 2 replicates were performed (2000 follicles in total), creating 2 data sets which differed only by the time of puncturing. The oocytes were given a score with regard to the morphology of the cumulus cell layers, Score 1 being oocytes surrounded by a compact cumulus (at least 2 intact cumulus cell layers), Score 2 being oocytes surrounded by an expanded cumulus, and Score 3 being the naked oocytes. Partially naked oocytes were given Score 1, when a maximum of one third of the total oocyte was naked. Otherwise Score 3 (naked oocyte) was given. Experiment 2 A different experimental protocol was used in Experiment 2 to investigate the net effect of the aspiration procedure on the recovery rate and the morphology of the COCs and to detect eventual oocyte losses or detrimental effects of the aspiration procedure on the oocytes. The COCs were previously obtained by slicing 116 slaughterhouse ovaries over a square Petri dish (Falcon 1012) filled with 10 cc Hepes buffered Talp solution (13) supplemented with 2.5 ml/lit Na-Heparin (25.000 IU/ml, Leo Pharmaceuticals, Vilvoorde, Belgium). The oocytes were searched for under a stereomicroscope. In total, 1000 oocytes surrounded by a compact cumulus and scored ‘compact cumulus oocyte complex’ (CCOC, Score I, Experiment 1) were divided into 20 groups of 50 CCOCs, spread over 3 replicates. Each of these groups was placed in a round Petri dish (Falcon 1008) filled with 3cc previously centrifuged (10 min, 3000 rad/min) follicular fluid, retrieved after puncturing an additional 26 slaughterhouse ovaries. After coating the needle and tubings with phosphate buffered saline (D-PBS, Gibco BRL, Life Technologies Ltd., Paisley, Scotland) supplemented with Na-Heparin (2.5 ml/ lit, 25.000 IU/ml, Leo Pharmaceuticals, Vilvoorde, Belgium) as used under in vivo conditions to prevent clotting, the CCOCs were aspirated by the system using 4 different types of disposable needles (Terumo neolus, Leuven, Belgium) at 5 different levels of aspiration vacuum (50, 70, 90, 110 and 130 mm Hg), thus creating the 20 different groups (needle type by level of aspiration vacuum). The CCOCs were placed in the Petri dish separated from each other to permit individual aspiration by the system. The following types of disposable needles with different diameters and bevels were used (Figure 1): Type 1, 18-gX2”, long bevelled, length 50 mm and external diameter 1.2 mm (18L); Type 2, 19-gX2”, long bevelled, length 50 mm and external diameter 1.1 mm (19L); Type 3, 20-gX2” long bevelled, length 50 mm and external diameter 0.9 mm (20L); and Type 4, 20-gX2”3/16

Theriogenology

1225

short bevelled, length 55 mm and external diameter 0.9 mm (20s). Types 3 and 4 are the same needles as used in Experiment 1. Aspiration was performed under visual control by means of a stereomicroscope to make sure that all 50 CCOCs in each group were actually aspirated (Figure 2). After aspiration, every group was re-collected separately in a round Petri dish (Falcon 1008) placed in an embryo filter (Em Con, Immuno Systems, Inc., Spring Valley, WI, USA) to finalize the identical set up as used for transvaginal OPU in vivo. The same needles, same connector, same length of tubing system and same way of collection were used, except for the fact that the

Figure 2. Experimental set up for Experiment 2: CCOCs are aspirated by the disposable system under visual control of a stereomicroscope and collected in a petridish placed in an embryo filter.

Theriogenology

CCOCs were collected immediately in the Petri dish and not on the filter membrane (Figure 2), the first being more practical for looking for the oocytes. After re-collection, the percentage of recovery was determined and the CCOCs were re-scored as previously described (Score 1, for oocytes still surrounded by a compact cumulus and Score 3, for naked or partially denuded oocytes). All COCs were taken up in an IVF system (23) to assess their developmental competence after the aspiration procedure. In Vitro Fertilization Following morphological evaluation, the oocytes were put into a loo-p1 drop of maturation medium, which consisted of TCM 199 bicarbonate supplemented with 20% estrous cow serum (ECS), 0.2 mM sodium pyruvate, 0.4 mM glutamine and 50 u&/ml gentamicin sulphate. This medium was covered with paraffin oil, and the COCs were incubated in 5% CO2 in humidified air at 39“C for 24 to 26 h. All treatment groups were matured, fertilized and cultured in separate drops. The oocytes were fertilized with frozen-thawed semen of a bull of known fertility and cultured following standard IVF procedures. Cleavage rate was determined 3 d post insemination. The number of expanded blastocysts 8 d post insemination was determined in relation to needle type, level of aspiration vacuum and COC morphology. Statistics Evaluation of the effect of needle tip or bevel on the oocyte recovery, cumulus oocyte complex morphology and subsequent developmental capacity of the oocytes after IVF was performed using the general linear model procedure (SPSS - Release 6.1 for Windows). Multiple logistic regression was used in a forward stepwise selection method with addition of the explanatory variables on condition that the probability of the difference in the likelihood for the reduced and full models is less than 0.05. Identical models were obtained with the backward selection method. These variables included the increase in aspiration vacuum level as a continuous variable, the type of needle tip bevel as categorical covariates and the interactions between increase in aspiration vacuum and type of needle tip bevel. The replication of the experiments yielded homogeneous results, which allowed pooling of the data. The applied model is as follows: In odds = In (rc/(l-7~)) = b,+b,x,+b,x,+b,,x,x, etc., with x, the increase in aspiration vacuum relative to 50 mm Hg and expressed per 20 mm Hg; x2 the indicator associated with the morphology of the oocyte (x2= 0 for compact cumulus oocyte complexes, x2= 1 for naked or partially denuded oocytes); xj, xq and x5 indicators associated with needle tip bevel or type. RESULTS Experiment

1

The results of Experiment I. in lvhich the influence of the needle bevel was studied. are given in Table 1 and Figure 3. The needle with the long bevel gave the highest recovery rate

Theriogenology

1227

Theriogenology

regardless of the aspiration vacuum used. The expected odds ratio of recovered oocytes for the 20L vs the 20s needle is e”.33= 1.40 (P< 0.001). For both types of needle tips, more oocytes are recovered at a higher aspiration vacuum with an odds ratio of e”.” = 1.12 per increase in vacuum pressure of 20 mm Hg (P < 0.001).

Compact Cumulus Oocytes

“O 4

0.8 -

v

20s

l

2OL

0.6 -

0.4 -

0.2 -

0.01

’

50

’

’

70

’

’

90

’

’

’

110

’

’

130

Aspiration Vacuum (mm Hg)

Figure 3. Multiple logistic regression of the proportion of CCOCs relative to the number of cumulus oocyte complexes recovered from punctured follicles as a function of the aspiration vacuum level for two 20-g needle bevels. Morphology was first analyzed relative to the actual number of recovered cumulus oocyte complexes. At low aspiration vacuum, most of the recovered oocytes are surrounded by a compact cumulus investment (an average of 64 % at 50 mm Hg for both needle types). This percentage decreased drastically with increasing aspiration vacuum. However, for the needle with the short bevel (2OS), this decrease is less pronounced (P d 0.001) as the odds for obtaining a CCOC decrease by 1.44 for short bevelled needles compared to 1.64 for long bevelled needles (Figure 3). As less oocytes are retrieved with a compact cumulus investment at higher aspiration

Theriogenology

1229

vacua, more naked oocytes are observed. When long bevelled needles were used, 75 % of the oocytes were naked, whereas this rate was 64 % for short bevelled needles, both at an aspiration vacuum

(Table

level of 130 mm Hg.

Second, morphology was expressed relative to the total number of punctured 1). At 50 mm Hg, an average for both needle types of 32 % of the punctured

follicles follicles

gave a CCOC and 10 % a naked or partially denuded oocyte. For both needle types, the odds for obtaining

a CCOC decrease

odds for obtaining Hg increase

by a factor of 1.29 (e” 258’) with increasing

naked oocytes

in aspiration

increase

vacuum (P < 0.001). The

by a factor of e045 = 1.57 and eo2* = 1.33 per 20 mm

vacuum (P < 0.001) when needles with a long, respectively

short bevel

are used. Finally,

developmental

after IVF. The prevalence at all aspiration follicles,

levels

capacity

of the retrieved

of blastocysts for both needle

remained

types. Expressed

an average of 9.7 % resulted in blastocysts

Experiment

of Experiment

and morphology

Application guidance

relative

complexes

was assessed

17 % of the recovered to the number

oocytes

of punctured

for both needle types at all aspiration

levels.

2

The results recovery

cumulus oocyte

approximately

of the aspiration system,

low aspiration

resulted

vacuum,

rate with an increasing

2, in which the net effect of the aspiration

of previously technique

increase

in aspiration

between

the 2 different

on previously

in an overall recovery

all needle types yielded aspiration vacuum

on the

scored CCOCs, using the disposable

of 79% of the aspirated similar recovery

vacuum was only observed

oocytes

20-g needles,

4 to 6. needle

(Figure 4). At

rates. An increase

in recovery

with the 18-g needle (odds ratio per

of 20 mm Hg = 1.22; P-c 0.05). The difference

bevelled

procedure

scored CCOCs was studied are given in Figures

as seen in the first experiment,

in recovery

rate

was not observed

here. At low aspiration cumulus

investment

thinner needles

vacuum, 82% of the recovered after aspiration.

Additionally,

oocytes were still surrounded this percentage

was higher

by a compact

when using the

(odds ratio of 1.57 referred to the 18-g needle; P < 0.01; Figure 5). For all needle

types a decreased yield of CCOCs was observed with increasing aspiration vacuum (a decrease in odds of 1.50 per increase in aspiration vacuum of 20 mm Hg; P < 0.001). All of the remaining ooytes

at low aspiration

aspiration needles,

pressures,

level (18%)

were naked.

As less CCOCs

the amount of naked oocytes increased

short and long bevelled,

were obtained

proportionally.

at higher

The 2 types of 20-g

gave similar results with respect to aspiration

pressure.

When morphology is expressed, relative to the initial amount of 50 CCOCs in each group (Figure 4), an average of 64% of the CCOCs remained surrounded by a compact cumulus and 14% became naked (at 50 mm Hg). For all needle types, the odds for obtaining a CCOC decrease by a factor of 1.30 (e026) with increasing aspiration vacuum (P < 0.001). The odds for obtaining

Theriogenology

1230

Morphology of oocyte complexes mm Hg

18L

50

70

90

110

130

18L

: 18-g long bevel

Legend

19L : 193 long bevel 20L : 20-g long bevel 20s

: 203

short bevel

Figure 4. Recovery and morphology different needle types at expressed as percentages addition of the number of

of previously scored CCOCs after aspiration with 4 5 different levels of aspiration vacuum. Results are relative to the number of aspirated CCOCs. The CCOCs and naked oocytes equals the recovery rate.

1231

Theriogenology

naked oocytes augment by a factor of eo.39= 1.48 per 20 mm Hg increase in aspiration vacuum (P + 0.001).

Compact Cumulus Oocytes ‘.OI

0.8 X 0.8

0.01

’

50

l

18L

v

19L. 2OL, 20s

’

s 70

’

’

90

’

’

’

110

’ 130

1

Aspiration Vacuum (mm Hg)

Figure 5. Multiple logistic regression of the proportion of CCOCs relative to the initial number of previously scored CCOCs as a function of the aspiration vacuum level for 4 needle types. Finally, developmental capacity of the retrieved cumulus oocyte complexes was assessed after IVF. Averaged over all needle types, the prevalence of blastocysts, relative to the recovered oocytes, decreased with higher aspiration pressure (odds ratio of 1.16 per 20 mm Hg, P < 0.05). Expressed relative to the initial number of previously scored CCOC in each group (Figure 6) on the average 12% of the CCOCs resulted in blastocysts, and no statistically significant effect of needle type or aspiration vacuum level on the final outcome in terms of blastocysts could be detected.

1232

Theriogenology

Blastocysts

Figure 6. Final outcome of blastocysts after aspiration of CCOCs with 4 different needle types at 5 levels of aspiration vacuum, relative to the number of aspirated CCOCs in each group.

DISCUSSION An increase in the quality and quantity of the COC retrieved with the transvaginal OPU technique in combination with a more economical approach can only be achieved after a profound study on the different aspects involved. Apart from biological factors such as hormonal pretreatment of the animals prior to puncture, there are more technical aspects dealing with the most reliable way to reach the follicles and retrieve the present oocytes. To eliminate the disadvantages of the long, non-disposable needles, a new oocyte pick-up device equipped with a disposable needle guidance system was constructed (2). Because of the use of disposables, an enormous amount of different needle types became available and in combination with the different aspiration vacua used, they can exert various effects on the morphology and therefore on the subsequent developmental capacity of the oocytes. In a previous study, we demonstrated by means of an in vitro oocyte pick-up simulation that a simple change in needle diameter in

1233

Theriogenology

combination

with an altered

Furthermore,

the thickest needle (18-g) yielded the highest recovery rate but also gave the highest

proportion

of naked oocytes.

rate, the proportion

aspiration

vacuum

Although

of oocytes

can affect the morphology

thinner needles

surrounded

(21-g) yielded

by a compact

cumulus

of the COC (4).

a lower oocyte

was higher.

recovery

For all needle

diameters, the proportion of oocytes surrounded by a compact cumulus decreased progressively as the vacuum increased. Based on these results, we recommended the use of thinner needles (19g, 21-g) at a moderate aspiration vacuum. An additional needle

aspect,

tip shape

Literature

which

or bevel

is studied

on oocyte

is very scarce concerning

needle bevels,

although

in our previous

(4), an in vitro simulation

First of all, the construction

and tubings

in exactly

separately, a sufficient

3 replicates. procedure,

without making mistakes

diameters

of the transvaginal

needles

oocyte

pick-up

as in vivo. Secondly,

can be a measure

due to insufficient

the actual puncture

needles.

of the follicle

of the aspiration

visualization,

more than once, which can happen in vivo.

the use of long and short bevelled

needle,

for the net effect

ultrasonal

missing

When a follicle

wall happens

by

the needle

tip

before

(RMWJ Baltussen,

anticipated follicle

experiment,

vacuum application

after the other. According

surrounded aspiration

by a compact vacuum.

which confirms short bevelled

cumulus

This percentage

our previous needle.

Contrasting

used. The possible of the aspiration

to the morphology, investment decreased

observations

This suggests

losses vacuum

described pressure

with increasing

this decrease

that a change to short bevelled

higher aspiration

pressure

lose the cumulus

cells along the long sharp ridge. In terms of final outcome

is used since the longer bevel constitutes

of the recovered

oocytes

at all aspiration

one

COCs were

needles

aspiration

at low vacuum,

is less pronounced needles

rate

are probably

when puncturing

for both long and short bevelled

(4). However,

recovery

earlier

most (64%) of the retrieved

drastically

fluid loss

results are obtained

where the needle with the long bevel gives the highest

of the aspiration by a constant

communication).

can

is punctured

as a whole. Leakage through the long bevel with oocyte and follicular personal

follicles

Several suppositions

enters the follicle in the present

was

this simulation

could be possible regardless

and

(1). As

set up assures the needle to be brought into each follicle

differences

one follicle

a long bevelled

COC.

with different

of the device permits the use of the needle

the same conditions

And finally, the simulation so the detected

be made concerning

of the

amount of oocytes to be retrieved within the same time period spread over

separately, or puncturing

needles

effect

of the retrieved

are made using long non-disposable

used for several reasons. guarantees

paper, is the possible

and the morphology

the use of disposable

similar comparisons

experiments

in the present

recovery

is indicated

for the when a

a higher risk for the COC to of blastocysts,

17%

vacua for both needle types are cultured to blastocysts

8 d post aspiration. As stated before complex pathway disposable needles

(2), one of the possible

disadvantages

of a disposable

system is the more

the oocyte has to follow from the follicle to the collecting tube. Using implicates the use of a connecting piece and longer tubings. Therefore, we

tried to isolate the net effect of the aspiration procedure on the recovery rate and morphology of a fixed number of previously scored CCOCs. These CCOCs were obtained after slicing

Theriogenology

1234

slaughterhouse ovaries instead of puncturing, because this first aspiration by needle and syringe would select already the CCOCs in which the cumulus investment is very strongly attached to the oocyte. CCOCs retrieved after slicing are not submitted to any kind of stress before the actual aspiration by the system and therefore mimic the in vivo situation in a better way. Our results show that on the average nearly 80% of the previously fixed number (50) of aspirated CCOCs in each group are re-retrieved after the aspiration procedure. This indicates a possible loss of oocytes in the system of 1 out of 5. Although comparable experiments have not been carried out with long, non-disposable needles, our results suggest that the use of disposables is highly effective for oocyte retrieval. Starting with COCs, previously scored as compact cumulus oocyte complexes (CCOC), the net effect of the aspiration procedure using different disposable needles at different aspiration vacua can be determined by re-scoring the CCOCs after being submitted to the aspiration procedure. In this sense, changes in morphology of the CCOCs are a measure for damage caused by aspiration. Using a low aspiration vacuum, 82% of the recovered oocytes were still surrounded by a compact cumulus investment after being aspirated. This percentage is even higher (86-90%) when using thinner needles and decreases proportionally for all needle types when aspiration vacuum increases, which confirms earlier observations (4). In other words, on the average 1 out of 5 to 1 out of 10 COCs of initial good quality (compact cumulus) is microscopically damaged by the aspiration procedure in a way that the cumulus cells are totally or partially ripped off, which seriously impairs the in vitro developmental potential of the oocyte (26). There were no significant differences between the two 20-g needles with different bevels. Although these results suggest once again that thinner needles cause less stress to the COC in terms of cumulus cell damage, it is much more difficult to observe these differences in the final outcome of blastocysts. One of the reasons for this is that the embryos are cultured for 7 d in the IVF system and are influenced undoubtedly by an innumerable amount of factors. Expressed relative to the recovered oocytes, the prevalence of blastocysts decreases with higher aspiration pressure, confirming previous results (4). In conclusion, our results show that the length of the needle bevel can have a significant effect on oocyte recovery, the long bevelled needle resulting in a higher oocyte recovery rate. As soon as higher aspiration vacua are used, the number of CCOCs decreases but less prominent for the short bevelled needle (20s) compared to the long bevelled one (20L). The final outcome in terms of blastocysts is similar for both needle types. Starting with a fixed number previously scored compact cumulus oocyte complexes, the disposable needle system suggests to be highly effective for oocyte retrieval, since nearly 80% of the aspirated CCOCs could be retrieved. Using a low aspiration vacuum and thin needles, up to 90% of these CCOCs withstand the aspiration procedure undamaged. Increasing the aspiration vacuum results in a decrease of the number of CCOCs,

which is less pronounced

when using thinner needles.

1235

Theriogenology

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