Dept. of Theriogenology.

Vet. Med. , Assiut Uni., Egypt

Quantity and quality of harvested buffalo oocytes  in relation to ovarian structures and methods of collection

(With 4 Tables)

By

A.Kh. Abdel-Razek and. A.M. Ali

کمية ونوعية البويضات المجموعة من مبايض الجاموس وعلاقتها بترکيبات المبيض وطريقة الجمع

عبد الرازق خليفة عبد الرازق ،  أحمد مصطفى علي

لدراسة تأثير ترکيبات المبيض على نوعية وکمية البويضات المتحصل عليها من مبايض الجاموس المذبوح لاستعمالها فى التلقيح خارج الرحم تم فى الجزء الأول من الدراسة جمع 40 مبيض وتقسيمهم الى أربعة مجموعات : تلک التى تحوى جريبة سائدة (14 مبيض), والتى تحوى جسم أصفر(9 مبايض) , والتى تحوى کلتا الترکيبتين (5 مبيض), والأخيرة کانت لا تحوى أى من الترکيبتين ( 12 مبيض). تم عد الجريبات السطحية والتى يتراوح قطرها بين 3-8 مم ثم تم سحب السائل الجريبى منها بواسطة محقن وفحصه مجهريا لجمع البويضات. تم عد البويضات المتحصل عليها وتقييمها طبقا لعدد وتماسک خلايا طبقات الرکام المحيطة بها وکذلک طبيعة السيتوبلازم. فى الجزء الثاني من الدراسة تم مقارنة عدد ونوعية البويضات التى تم الحصول عليها فى التجربة السابقة (طريقة السحب بالمحقن) بتلک التى تم الحصول عليها بتقطيع المبيض الى اجزاء صغيرة بواسطة سلاح مشرط ثم غسل  تلک الأجزاء فى محلول فسيولوجى ((PBS. تم فحص ذلک المحلول لجمع البويضات وتقييمها. وقد وجد من النتائج ان عدد البويضات التى تم الحصول عليها من المبايض التى تحمل جريبة سائدة کان اقل فى العدد وکذلک کانت مواصفات البويضات اقل فى جودتها من تلک التى جمعت من المجموعات الأخرى. کما وجد أيضا ان عدد البويضات التى تم الحصول عليها بتقطيع المبيض کانت اکثر فى العدد وذات جودة عالية عن تلک التى تم الحصول عليها بواسطة سحب السائل الجريبى.

SUMMARY

A total number of 68 buffalo-cows ovaries were collected from slaughterhouse. In the first part of this study, 40 ovaries were classified according to the ovarian structures into: those with dominant follicle (DF, n = 14) , with corpus luteum  (CL, n=9 ), with DF and CL  (n=5) and those without DF or CL (no dominant structure: nDS, n = 12). Surface follicles between 3 and 8 mm were enumerated and aspirated. The aspirated follicular fluids were examined microscopically for the oocytes. The recovered oocytes were counted and evaluated according to number and quality of the cumulus layers and character of the ooplasm. In the second part of the study, the obtained oocytes were compared with those obtained by slicing technique applied on 28 ovaries. The results revealed that, the average number of aspirated follicles/ovary was higher from ovaries with nDS (7.33 ± 3.8), than from ovaries with DF (3.67±2.2), p<0.5. Also, the average number of recovered oocytes/ovary was higher from ovaries with nDS  (5.75 ± 2.4), than from those with DF (2.79 ± 2), p<0.05. Ovaries which carried CL showed in-between results (4.0±3.7 aspirated follicles and 3.22±3.1 recovered oocytes). Higher number of oocytes with more than 5 cumulus layers was collected from ovaries with nDS (28.9%) than from ovaries with DF (5.1%), p<0.05.  Slicing technique increased significantly the average number of recovered oocytes/ovary than the aspiration technique, (5.00 ± 2.3 vs 3.90±2.4) and the number of oocytes with more than 5 cumulus layers (62.1% vs 15.3%) p<0.05. It is concluded that 1) DF adversely affected the number and quality of recovered oocytes in buffalo, 2) ovarian slicing technique increased significantly the quantity and quality of the harvested oocytes.  

Key words: Buffalo, oocyte, dominant follicle, cumulus.

Introduction

Nowadays, there is a considerable interest in developing the technology of in-vitro-fertilization (IVF) for embryo production in buffaloes (Kruel, 1991 and Gordon, 1997). However, in comparison to cattle and camels, buffaloes produced lower number and less culturable oocytes (Ali and Abdel-Razek, 2001). The number and quality of the oocytes may be affected by the method of oocyte collection as well as by the structures present on the ovary (Kumar et al., 1997 and Ali et al., 1999). As the oocyte grows within the follicles, a number of factors may  influence its health and developmental competence (Abdoon and Kandil, 2001). These factors include stage of estrous cycle, follicle size and the predominant ovarian structure (Hagemann et al., 1999).

Although, dominant follicle (DF) has been proved to adversely affect the developmental competence of the oocytes in cattle (Smith et al., 1996), little is known about its effect in buffaloes. Like cattle, follicles in buffalo develop in a wave like pattern, with two to three waves per a cycle (Ali et al., 2003). Each wave is characterized by recruitment of many small follicles, followed by selection of one of them (DF) and regression of the others (subordinate follicles) (Taneja et al., 1996). In cattle, there is evidence that the DF suppress the development of the subordinate follicles.

The aim of the current study was to: 1) Clarify the effect of the presence of DF on the number and quality of the oocyte obtained from subordinate follicles in buffalo-cows. 2) Compare between the aspiration and slicing techniques on the quantity and quality of harvested oocytes.

Materials and Methods

Female reproductive tracts of native buffalo-cows were collected from local slaughterhouse shortly after evisceration. The ovaries (n = 68) were dissected from the tracts and transferred in a previously warmed thermos (35 ^o C) within about one hour to the laboratory.

Part 1

In the laboratory, a number of 40 ovaries were  classified according to the predominant ovarian structure into those with DF (defined as that which reach the diameter of 8 mm or more and exceed the diameter of other follicles on the ovary) (DF, n = 14) , those with corpus luteum  (CL, n=9 ), those with DF and CL (DF &CL, n=5) and those without DF or CL (no dominant structures: nDS, n = 12). Subordinate follicles between 3 and 8 mm in diameter were enumerated and aspirated from  groups. Aspiration of follicles was performed using 19 G needle attached to 5 ml syringe. The aspirated follicular fluids were collected in conical glass centrifuge tubes. After about 15 minutes, the sediment were aspirated with pipette and poured in small Petri dishes, diluted with dulbeccos phosphate buffer saline (PBS) supplemented with 3mg/ml bovine serum albumin (BSA) and examined under stereomicroscope. The recovered oocytes were counted and evaluated according to the number of cumulus cell layers (< 3, 3-5 and > 5 layers), compactness of cumulus cells (compact or expanded) and homogeneity of the ooplasm (homogenous or heterogeneous) according to criteria used by  Ali and Abdel-Razek (2001).

Part II

A second group (n = 28) of ovaries were sliced in a Petri dish (7.5 cm in diameter) containing buffer solution (PBS and BSA). The slicing process was performed with sterile, clean and sharp scalpel. The sliced ovarian tissues were thoroughly washed in the buffer solution. The buffer solution then examined under stereomicroscope for the presence of oocytes (Sharma and Taneja, 2000). Oocytes were evaluated as in experiment I. The number and quality of the obtained oocytes were compared with those obtained in the first part.

Data are expressed in Mean ± SD. The Data were statistically analyzed using ANOVA-test to compare between means of the recovered oocytes during the different estrous cycle stages. CHI-test was used to compare between the percentages of the oocyte quality, while t-test was used to compare between the two recovery methods ( SAS, 1992).

Results

The number of aspirated follicles and oocyte recovered  from ovaries with  DF, CL, DF&CL and with nDS are illustrated in table 1. More follicles were aspirated from ovaries with nDS (mean of 7.33±3.8 follicles/ovary) than from those with DF, CL and CL&DF (mean of 3.67±2.2, 4±3.7 and 4.8±2.3 follicles/ovary, respectively), p< 0.05.

Also, a higher number of oocytes were obtained from ovaries with nDS (average of 5.75±24 oocyte/ovary) than from those with DF, CL and CL&DF (average of 2.79±2.0, 3.22±3.1 and 3.8±1.9 oocytes/ovary),  p< 0.05.

Effect of the predominant ovarian structures on the quality of the recovered oocytes is showed in Table 2. Higher number of oocytes with more than 5 cumulus layers was collected from ovaries with nDS (28.9%) than from ovaries with DF (5.1%), p< 0.05. However, there was no difference between the oocytes from ovaries without or with DF and/or CL with respect to the compactness of the cumulus or the homogeneity of the ooplasm.

Effect of the recovery method (aspiration vs. slicing) on the number of the harvested oocytes is shown in Table 3. The slicing technique increased significantly (p< 0.05) the recovered oocytes (mean of 5.00±2.3 oocyte/ovary) than the aspiration method (mean of 3.90±2.4 oocytes/ovary).

Moreover, slicing technique increased significantly (p< 0.05) the number of oocytes with more than 5 cumulus layers (62.1%) than the aspiration technique (15.3%) (Table 4). The incidences of harvesting denuded oocytes or those with less than 3 cumulus layers as well as those with 3-5 cumulus layers were significantly higher (p < 0.05) in the aspiration method than in the slicing technique (Table 4). However, there was no difference between the two recovery methods in concerning to the compactness of the cumulus layer or the homogeneity of the ooplasm.    

Table 1: Effect of ovarian structures on the number of aspirated follicles and the recovered oocytes

Values in Means±SD. CL: corpus luteum, DF: dominant follicle, nDS: no dominant structure (no DF or CL). Values with different superscript letters in the same row differ significantly (p<0.05).   

Table 2: Effect of ovarian structures on the quality of the recovered oocytes

CL: corpus luteum, DF: dominant follicle, nDS: no dominant structure ( no DF nor CL)

Percentages with different superscript letters in the same row differ significantly (p<0.05).   

Table 3: Effect of the recovery methods on the number of the recovered oocytes.

Values in Mean±SD. Means with different superscript letters in the same row differ significantly (p<0.05).   

Table 4: Effect of the recovery method on the quality of the recovered oocytes

Percentages with different superscript letters in the same row differ significantly (p<0.05).   

Discussion

Although non-surgical method of embryo recovery from superovulated donor buffaloes has been available for some years, such method suffer from the problem of low superovulatory response (Madan, 1990, Misra et al., 1990). With the appropriate IVF system, ovaries from slaughterhouse may provide an abundant and easily accessible source of buffalo embryos for breed improvement.

The mammalian oocyte grows and gains developmental competence within a complex of follicular and ovarian environment. As the oocyte grows within the follicles a number of factors might influence its health and developmental competence (Hageman, 1999). Little is known about the effect of DF on the oocytes of the subordinate follicles in buffaloes. This study was planned to investigate the influence of DF on the quantity and quality of the subordinate follicles in buffalo-cows ovaries.

In the current study more follicles were aspirated from ovaries without DF than that from those with DF. Consequently, a higher number of oocytes were obtained from ovaries without DF than that with DF. This indicated a negative effect of the DF on the quantity of the subordinate follicles of the same wave. The same observation was noticed in cattle using ultrasound examination, where the number of medium sized follicles (5–8 mm in diameter) was lower on ovaries with DF than on the contralateral one (Fortune, 1994, Kastelic, 1994 and Ali, 2000). Daily ultrasound examination revealed that, at the beginning of estrous cycle (day of ovulation) a number of small follicles (< 3 mm) recurred. These follicles developed together up to day 4 or 5 of the cycle, where thereafter only one follicle continued to develop (DF), while the other follicles (subordinate follicles) stop growth and reduced in number (Singh et al, 2000 Ali, 2000, Ginther  et al., 1996, Ireland et al., 1979)

The presence of DF not only affect the growth of subordinate follicles but the study revealed the tremendous effect on the oocyte morphology, where higher number of oocytes with more than 5 cumulus layers was collected from ovaries with nDF than from those with DF. This indicates a negative effect of DF on the quality of the oocyte from the subordinate follicles. In cattle, some authors reported a negative effect (Matton et al., 1981 and Wolfsdorf et al., 1997), others could not find any adverse influence (Smith et al., 1996).

The question raised now, how the DF adversely affects the oocyte from the subordinate follicles. There are two possibilities. The first one is through the production of estrogen and inhibin hormones from the DF. Estrogen and inhibin hormones suppress the release of FSH from the anterior pituitary. Because, FSH is essential for the development of the subordinate follicles, so suppression of FSH results in atresia and degeneration of the subordinate follicles (Danell, 1987). Draincourt (1991) named this possibility as the negative or systemic way of follicular suppression. There are some observations, which could support this hypothesis. Destruction or removal of the DF in cattle resulted in increase blood level of FSH and LH one day after DF removal (Ginther et al., 1998, Adams et al., 1992 Villa-Godoy et al., 1985). A new follicular growth started 2 days after destruction of the DF (Staigmiller and England, 1985, Ko et al., 1991, Bergfelt et al., 1991, Adams et al., 1992). Immunization against Inhibin hormone increased the ovulation rate in heifers (Morris, et al., 1993). The second possibility of the effect of DF on the subordinate ones is the direct or active way, where the DF releases certain substances (e.g. IGF, EGE), which affected locally and directly (paracrine effect) on the subordinate follicles. Such substances decrease the sensitivity of the subordinate follicles for the FSH (Draincourt, 1991).

In contrast to Abdoon and Kandil (2001), who recorded that the presence of CL stimulates the development of a significantly higher number ovarian follicles which produced a significantly higher number of good quality oocytes, the present study revealed that, neither CL nor DF  improve the number and quality of the obtained oocyte. This agree with the result of El Sherry (2003). While absence of the two structures improve the quantity and quality of the harvested oocytes. A similar results recorded by De Wit et al., (2000), that recorded the stage of the cycle had no effect on the distribution of the COC.   

The effects of  method of harvesting of oocyte were studied  and it was clear that slicing the ovaries produced more oocytes than the aspiration technique. This can be explained by: aspiration harvest the oocyte population from only the visible surface follicles. However slicing the ovaries make collection of oocytes  possible from surface as well as cortical follicles. When the number of oocytes from both peripheral and cortical follicles combined, the yield of oocytes was approximately doubled (Arlotto et al., 1996 and Sharma and Taneja, 2000).

The study also revealed that, oocytes harvested by slicing posses the culturable  and competing quality than that collected by aspiration. Where the slicing technique produces less number of denuded oocytes and more with >5 comulus layers which is also more compact than that collected by aspiration. This is in agreement with the result of Das et al., (1996), Kumar et al., (1997), and Datta and Goswami, (1998). This may be due to the effect of needle diameter or the aspiration pressure              (Hashimoto et al., 1999). But this disagreed with the finding of Abddoon and Kandil (2001).

It can finally be concluded that, presence of dominant follicle on the buffalo ovary affect the number and quality of harvested oocytes. Slicing of ovaries is a preferable method for harvesting more oocytes capable to compete during in vitro maturation and fertilization.

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