Dept. of Theriogenology,

Fac. of Vet. Med., Assiut University, Assiut Egypt

EFFECT OF SEASONS ON OVARIAN MORPHOLOGY  AND OOCYTES QUALITY IN SLAUGHTERED  BUFFALOES

(With  8 Tables)

By

A.M. OSMAN and S.H. SHEHATA

(Received at 5/3/2005)

على وصف المبيض ونوع البويضات فى الجاموس المذبوح    تأثيرالمواسم

احمد ممدوح حامد عثمان ,  شحاته حسن محمد شحاته

تم فحص وتشريح الاعضاء التناسليه لعدد 293 جاموسه سليمه وغير عشار مذبوحه فى السلخانه وتم الفحص بعد الذبح مباشرة فى المعمل لمدة عام کامل -وبعد استبعاد الاعضاء التناسليه الغير طبيعيه ترکزت الدراسه على 242 جهاز تناسلى سليم حيث تم فحص المبايض لکل جاموسه لمعرفة الحاله التناسليه وبعد ذلک تم قياس ابعاد کل مبيض وکل جسم اصفر وتم تقدير الوزن. تم عد الحويصلات الموجوده على سطح المبيض لکل جاموسه مع قياس قطر کل حويصله بالمسماک. بواسطة مضخه شفط کهربائيه وحقنه طبيه سعة 18    - متصله بقنينه زجاجيه محکمة الغطاء تم سحب محتويات الحويصلات. ومن خلال الاستريوميکروسکوب والميکروسکوب المعملى تم التعرف على البويضات الموجوده حيث تم عدها وفحصها لدراسة تأثير المواسم على جودتها. بينت النتائج ان المبايض التى تحمل جسم اصفر کانت اکبر من المبايض الاخرى بفروق معنويه (> 0.05 P) يمثل الجسم الاصفر 81 , . ± 1,30% من وزن المبيض. لم يکن هناک تأثير معنوى للمواسم علىمقاسات وأوزان المبيض. تبين ان عدد الحويصلات الکلية  بکل مبيض کان عاليا خلال موسم الربيع عن باقى المواسم (15,2 مقابل 9,1 فى الصيف، 8,1فى الخريف، 5,1 فى الشتاء) اما عدد الحويصلات الکبيره (أکبر من 9,0سم) بکل مبيض کان  قليلا جدا فى موسم الصيف عن باقى المواسم (27,0مقابل 43,0فى الربيع ،42,0فى الخريف ،37,0فى الشتاء). ادت الحويصلات الصغيره(أصغر من  4,. سم) فى المبيض خلال الصيف عنه فى باقى المواسم وکان الفرق معنويا بين الصيف والشتاء (0.05 > P). وکانت النسبة 6, 57 % فى الصيف مقابل 5,32 % فى الخريف و 8, 31 % فى الشتاء و 9, 42 % فى الربيع. بلغ العدد الاجمالى للبويضات المعرفه 563 بويضه منهم 477 بويضه سليمه (جيده وعليها تراکم من 3 طبقات) وکانوا اکثر انتشارا فى الحويصلات الکبيره عن 3, . سم - بلغت نسبة استخلاص البويضات الجيدة 6, . 9 % فى الحويصلات المشفطه ألتى کانت أکبر من 3 , . سم مقابل 5, 62 % فى الحويصلات الأصغر من ذلک. وکان تأثير المواسم على هذه النتائج يرجع الى توزيع الحويصلات الصغيرة فى المبايض خلال المواسم حيث کان مرتفعا خلال الصيف. بالنسبة للحويصلات الکبيرة فکانت أکثر انتشارا فى الربيع. کانت النسبة الکلية لاستخلاص البويضات هى 2, 90 % من اجمالي 624 حويصلة مشفطة. يستنتج من هذه الدراسة أن موسم الصيف ليس بالموسم المناسب لمثل هذه الدراسه نظرا لقلة عدد الحويصلات المناسبة فى الحجم والتى تعطى بويضات جيده سليمه تؤدى الى نجاح أى دراسة لاحقة. کما أن حرارة الجو المرتفع وسوء معدلات التغذية لها تأثير سلبى على الوظائف الفسيولوجية للمبايض خلال هذا الموسم فى الجاموس لانتاج حوصلات متوسطه وکبيرة ذات بويضات سليمة صالحة لأى تجارب لاحقة.

SUMMARY

Normal genitalia (242 out of 293) from healthy, non-pregnant slaughtered buffaloes were taken freshly and dissected. Ovaries were examined to determine the reproductive status of animals. Dimensions and weight of each ovary and Corpus Luteum (CL) were measured.  Number of follicles per animal was counted and their diameter was measured, and then follicular fluid was aspirated using an 18-gauge needle, fitted with stopper glass bottle and electric suction pump. Pooled samples were examined under a stereomicroscope to identify and evaluate the oocytes. The results showed that ovaries with CL were significantly larger (P<0.01). CL represents 30.1 ± 0.81% of ovarian weight. Seasons have no significant effect on both criteria. Total number of follicles per ovary was higher during spring   than other seasons (2.15 versus 1.9 summer, 1.8 autumn and 1.5 winter). Numbers of follicles (>0.9 cm) per ovary were much lower in summer than other seasons (0.27 versus 0.43 spring, 0.42 autumn and 0.37 winter). Total number of recovered oocytes was 563, and among them 477 was intact (good with comulus 3-4 layers). The incidence of intact oocytes was higher in medium and large follicles than smaller ones ( 92.3 % , 88.9 % and 71.8 % respectively. The average recovery rate was 90.2% from 624 aspirated follicles without marked variations between seasons. It was suggested that summer seemed to be unfavorable for similar studies due to the lower number of medium and large follicles having high incidence of intact oocytes leading to further successful  studies.

Key words: Buffalo, season, ovary, oocytes, follicles

INTRODUCTION

            The technology of in vitro fertilization still facing a lot of difficulties in buffaloes .The origin of the oocytes is of critical value in the progress  of such technique. Badr et al (2003) concluded that in order to achieve a reasonable recovery  rates for in vitro maturation (IVM) and fertilization (IVF) the ovaries might be stored not more than 4 hours post-slaughter in worm saline. In cows and buffaloes, Ectors et al (1995) and Yousaf and Chohan (2003) cited that oocytes from abattoir ovaries displayed a size-dependant ability to undergo maturation and oocytes larger than 4 mm can give better results for IVM and IVF. In addition, Neglia et al mentioned that aspirated cattle oocytes had significant higher cleavage rate than buffalo ( 83,9 % versus 64.8% )

            Abdoon and Kandil (2001) found that slicing of ovary produced higher oocytes recovery rate than aspiration but percentages of fair, poor and denuded oocytes were also higher. Sharma and Taneja (2000) recorded that individual follicle isolation yielded the highest number of oocytes per ovary when compared with other methods. Moreover, the presence of corpora lutea significantly raised the number of follicles and number of good quality oocytes. However, good quality oocytes were recovered from ovaries of cycling buffaloes at days 1-3 and 10-16 of the estrous cycle (Abdoon and Kandil, 2001).

            Suzuki et al (1992) and Abdoon et al (2001) reported that buffalo oocytes surrounded by multi-layers of cumulus cells had a significant higher rates for IVM and IVF than partially surrounded or denuded oocytes. The cumulus cells surrounding the oocytes are essential to facilitate the transport of nutrients and signals into and out of the oocytes (Anderson 1991).

            In order to optimize the IVF technology in buffalo, Ravindranatha et al (2003) suggested that storage of ovaries at 4 C° for 12 or 24 hour significantly reduced the developmental potential of oocytes. Kumar et al (1997) established a biphasic relationship of growth between oocytes diameter and follicular size in ovaries from slaughtered buffaloes. It was concluded by Raghu et al (2002) that the larger the size of the follicles the greater the in vitro development  and competence of buffalo oocytes.

            The body condition score (3-5 degrees) of buffalo was recorded by Ahmed et al (1999) to improve the oocytes quality. Nandi et al  (2001) found that more oocytes were recovered per ovary during the cool period of the year than during the hot period in Indian buffaloes. Reproductive efficiency in buffalo cows is hampered by many factors including the season. Osman (1984), El Naggar et al (1985), Abdoon et al (1992), Srivastava and Pant (1999) and Sing et al (2000) gave an ample information about the domination of ovarian sub-function in water buffalo during the dry hot period of the year.

            The current investigation was carried out to emphasize  the effect of seasons on ovarian morphology as implicated to the quality of oocytes in slaughtered buffaloes.

MATERIALS and METHODS

Collection of genitalia:

From a local slaughterhouse, 293 genitalia were taken freshly from adult healthy buffalo cows and immediately brought to the lab within an hour (20 km distance). The materials were collected during a whole year. Abnormal, pathologically affected genitalia were excluded and the study included finally 242  pair ovaries out of them 3 contained large dermoid cysts. Further 10 ovaries with corpora lutea were taken from pregnant genitalia (less than 2 months) for comparative studies.

Thorough morphological examinations were done to classify the reproductive condition of  the animal into: proestrus, estrus, metestrus and diestrus according to the ovarian structures. Intact ovaries were taken, washed with water and kept in a dish with normal saline. Dimensions and weights of each ovary and corpus lutea were performed.  Numbers of follicles were counted per each ovary and per animal. Their diameters were measured  to the nearest 1/10 cm using a caliber.

Follicular fluid aspiration:

         Ovaries with follicles were plotted with filter paper. An electrical suction pump, fitted with a sterile stopper glass bottle and a receiving sterile conical flask connected to a rubber tube with 18 gauge needle  were used to puncture and aspirate the follicular fluid. At the end of each collection, few ml of saline were drawn to avoid any oocytes losses. Pool samples in the flask were transferred to a sterile test tube and kept vertically for 20 minutes before microscopic examination. Attention was paid to collect the pool samples according to the size of the follicles in       many cases.

Oocyte-Cumulus Complexes (OCCs) recovery and evaluation:

Using a lab water pump the supernatant fluid in the test tube was removed. The rest portion containing the oocytes was transferred to small petri dishes and examined under a Stereomicroscope to identify, count and evaluate the oocytes  according to Yong and Lu (1990). Some oocytes were aspirated with a mouth pipette, fixed on slide and stained with aceto-orcine and Gemsa stains for further investigations.

The results were recorded as:

  Intact good quality oocytes:

            With cumulus > 2 layers and  homogenous cytoplasm  

  Denuded bad quality oocytes:

            With cumulus < 3 layers.

Statistical analysis:

All data were subjected for statistical analysis according to Snedecor and Cochran (1980) and the Costat Computer Program (1986).

RESULTS

            The biometrical studies of the ovaries from slaughtered buffaloes as related to seasons are presented in Tables 1-4. It is evident from these findings that no significant differences could be traced in the dimensions and weights of the ovaries between seasons as well as between the right and left side. The 3 ovaries with dermoid cysts were much larger in size and varied in weight between 8-12 gm and excluded from the data. 

            On the other hand significant differences were proved between the different conditions of the ovaries within seasons and as well among all organs (Tables 5). Ovaries carrying corpora lutea and follicles were the largest while static ovaries were the smallest. Ovaries with corpora lulea alone or follicles alone lies in between with regard to dimensions (Length, breadth and thickness) and weight.

            Table 6 summarized the weights of corpora lutea as related to their ovaries. Significant differences between seasons were not evident and the average corpus luteum weight represents about 1/3 of the intact ovary. Moreover, the corpus luteum periodicum was significantly (P<0.05) smaller than that of pregnancy (1.59± 0.04, n= 158, versus  2.12 ± 0.2 gm, n=10).

            As far as the numbers of follicles  per ovaries are concerned, table 7 showed their results according to their diameters in different seasons.

            There is no marked difference between the right and left ovaries. However, during summer the percentages of large follicles (>0.9 cm) represented only 16% of the total follicles while during autumn, winter and spring the percentages were 31%, 32% and 26% respectively. Per ovary the average number of large follicles (> 0.9 cm) was low in summer 0.27, versus  0.44 spring  0.42 autumn and 0.37 winter.

The number of small follicles (< 0. 4 cm) per ovary was higher  in summer than any other seasons (0.9 summer versus o.32 autumn, 0.24 winter and 0.6 spring). It is evident from these findings that during summer the incidence of large follicles per ovary decreased while that of small follicles increased. Moreover, the incidence of ovaries without CL and or follicles (static ovaries) was relatively higher in summer than other seasons (22.9 % summer, versus 10.8 % autumn, 20.8 spring and 18.9 % winter.

  The total number of follicles per ovary (Table 8) was higher during spring than any other seasons. The relatively high incidence for the number of follicles per ovary during summer (1.94) was attributed to the high incidence of small dormant follicles (< 0.4 cm) per ovary in that season (57.6 % versus 32.5% autumn, 31.8% winter and 42.9%  spring).

The intact good quality oocytes of buffaloes were surrounded by variable thickness of cumulus, more than 3 layers, within a condense homogenous cytoplasm. Oocytes surrounded by less layers or dispersed granulosa cells were considered denuded and of bad quality. Degenerated or abnormal shaped oocytes were among this group. The average recovery rate for all oocytes (Table 8) was 90.4 %  from 624 aspirated follicles (77.1 % for intact and 13,3 % for denuded oocytes). Moreover, the average number of oocytes per ovary was 1.68. The recovered total oocytes per ovary was higher in spring followed by summer, autumn and winter but the differences were not significant.

            Regarding the effect of follicular size on oocytes quality, 63 intact oocytes were recovered from 76 follicles with variable size. The incidence of good quality oocytes was 92.3 % in medium size follicle (0.4-0.9 cm ) versus 88.9 %  in larger  and 71.8 % in smaller follicles.

DISCUSSION

            The low efficiency of superovulation and embryo transfer program in  buffalo lead to an increasing interest for in vitro embryo production technologies to faster the propagation of superior germ plasm in this species (Gasparrini, 2003). The available literature reviewing an ample evidence about the factors influencing the quality of oocytes obtained from abattoir for such purposes. The integral effect of season on in vitro oocytes maturation and fertilization appeared lacking and needs certain elucidation.

            The obtained results showed variations between seasons in the biometrical data of the ovaries but not at a significant levels. The prevalence of ovaries without CL and F in summer coincides with the recent investigation of Ahmed (1997). In harmony with these findings, Hemieda (1988) and Eltohamy (1994) cited that ovarian inactivity during the dry season of the year (June-November) was high. However, the significant differences observed in weight between the different cyclic conditions of the ovaries, support the findings of Farrag (1978) and Osman (1984) that ovaries with CL and F were larger than ovaries without any of both . The significant difference between CL grvidatis and CL periodicum weights might reflect the huge tasks for progesterone production during early pregnancy in buffaloes. Between seasons, no valuable differences could be traced in CL weight as related to the ovarian weight. However, Abdoon et al (1992) recorded higher levels of progesterone during the luteal phase of the cycle in summer, autumn and winter than spring. 

            It is concluded from the biometrical investigations on buffalo ovaries that seasons have no significant effects on their size or weight.

            In concern with the number and diameter of follicles per ovary, the obtained results showed marked reduction in the incidence of large follicles (> 0.9 cm) during summer (0.27 versus 0.38 in other seasons). In addition, the increased number of small follicles ( < 0.4 cm ) per ovary in summer than other seasons (0.9 versus 0.38) leads us to the persuasion that inactive ovaries were dominated in such dry hot season in Egypt. Singh et al (2000) cited that reproductive efficiency is hampered in Indian buffalo by poor estrus expression in summer.     

            Ahmed (1997) reported that ovarian inactivity was prevailed during the dry season of the year and in animals with poor body condition score compared to green season and those with good body condition score.

            With regard to the incidence of intact good quality oocytes per ovary, our results showed no significant effects for the seasons on such criteria. On the other hand, the incidence of intact oocytes was higher (92.3 %) in the medium sized follicles (0.4-0.9 cm) than either the larger (88.9 %) or smaller follicles (71.8 %). Ectors et al (1995) observed that better in vitro development occurred with oocytes collected from follicles larger than 4 mm. Comparable results were recorded by Kumar et al (1997), Yousaf and Chohan (2003) who emphasized a size dependent ability between follicles and oocytes to obtain better results for IVF.

            As a major conclusion from the present study, it seems possible that summer can adversely affect the functional activity of the ovaries  to produce medium and large size follicles suitable to give high incidence of good quality oocytes favorable for further successful IVF processing. Thus, dry summer season might be not suitable for such technique of breeding in buffalo.

References

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Table 1: Biometry of the ovaries in slaughtered buffaloes during Spring.

Table 2: Biometry of the ovaries in slaughtered buffaloes during Summer.

Table 3: Biometry of the ovaries in slaughtered buffaloes during Autumn.

Table 4: Biometry of the ovaries in slaughtered buffaloes during Winter.

Table 5: Biometry of the ovaries in slaughtered buffaloes as related to their conditions among all samples.

Table 6: Weights of Corpora Lutea as related to their ovaries among different seasons in slaughtered buffaloes.