Department of Animal Production, Faculty of Agriculture,

Alexandria University, Alexandria, Egypt.



Five hundred pairs of buffalo ovaries were collected from a local abattoir and transported properly to the laboratory. Ovaries were classified according to their pathological and physiological status into 3 categories, normal, cystic (largest follicle diameter > 20mm with turbid appearance, or cystic corpus luteum with a fluid filled cavity) and double ovulated ovaries (one or both ovaries containing 2 CLs). Follicles were classified into large (>8-20mm), medium (4-8mm) and small (<4mm). Within each physiological category, follicular fluids were aspirated individually for each size of follicles per pairs of ovaries. Follicular fluids were analyzed for acid and alkaline phosphatase (AcP and AlP), alanine aminotransferase (ALT) and asparatate amino transferase (AST), free radicals, total proteins, albumin and progesterone levels.

Fluids of largest follicle of double ovulated ovaries contained higher enzymatic activities compared with optic and normal ovaries. Thus, the levels of AlP for the three groups were 783vs 147.2 and 112.5 IU/L, AcP values were 82.1 vs 1.2 and 2.6 IU/L, ALT values were  35.7 vs 19.6 and 21.0 IU/L and AST values were 38.0 vs 23.4 and 21.7 IU/L. Moreover, progesterone concentrations in double ovulated (5.5ng/ml) and  normal (5.3 ng/ml) were higher than cystic (4.7 ng/ml) ovaries. Concomitantly, total protein and albumin concentrations were higher in double ovulated (403.3 mg/ml and 3.6 g/dL) than cystic (283.4 mg/ml and 2.4g/dL) and  normal ovaries (233.9 mg/ml and 2.1 g/dL) ovaries, respectively. Contrariwise, free radicals concentration was higher in cystic (0.50 nmol/ ml) than in double (0.06 nmol/ml) and normal (0.08 nmol/ml) ovaries.

Keywords: Buffalo, ovary, follicular fluids, progesterone, enzymes.



Buffaloes play a prominent role in the Egyptian livestock production and in turn in national economy. Regarding meat production, an estimated 1.6 million metric tons of buffalo meat is produced annually worldwide (Agarwal and Tomar, 1998). Of this about 231000 metric tons are produced in Egypt (FAO, Production Yearbook, 1998). The slaughter of female buffalo and cattle is subjected to a regulatory law which approve slaughtering females at 5 years of age regardless of their reproductive capacity.

Follicular fluids of ovaries play an important role in the physiology of follicular growth, oocyte maturation and ovulation (Fortune, 1994). Although the factors that regulate follicular development have not been fully elucidated. Follicular fluid is probably not a simple transudate of blood, rather a complex of restricted components of serum and follicular synthesized secretions (Bellin and Ax, 1984). Thus, there is a need to understand the basic biochemical and endocrinologic changes that accompany follicular maturation in buffalo and to correlate these with the morphological characteristics. The present study was designed to monitor a number of metabolic and hormonal characteristics in order to characterize follicular development and fertility status of the slaughtered female buffaloes. Also, variations between normal, cystic and double ovulated ovaries were concerned.



Ovarian collection and evaluation :

Ovaries were freshly collected from slaughtered female introduced to the abattoir as above 5 yearsold . A sanitary procedure was applied to collect ovaries in chilled saline solution (0.9% NaCl). Follicles were classified into three major categories; (a) Normal: which have follicular structures with  one corpus luteum (CL) on one ovary (right or left ovary); (b) Cystic: one or both ovaries contained large follicle of more than 20mm diameter with opaque liquid appearance and thick follicular wall, or contained a fluid-filled cystic CL; (c) Double ovulated : one or both ovaries contained two well-developed corpora lutea. Follicles of each pairs of ovaries were classified according to their diameter into; large (>8-20mm), medium (4-8mm) and small (<4mm). Also, the different population of follicles in each pair of ovaries were recorded (i.e. some ovaries contained small, medium and large and others contained small and large follicle only … etc.).


Follicular fluids aspiration:

Fluids were collected using 1CC syringes with 20-gauge needles for small and medium follicular fluid aspiration, however 3CC syringes with 18-gauge needles were used to collect fluids of large or cystic follicles. Fluids of each category of follicles in each pair of ovaries were pooled. Fluids were centrifuged (3000 rpm/15 minutes) and supernatant was decanted and frozen (-20oC) until used for assays.


Analysis of follicular fluids :

Total protein concentration was determined by the method of Lowry et al.  (1951).

Albumin concentrations were determined by the method of Doumas et al. (1977).

Free radicals, thiobarbituric acid- reactive substances (TBARS) were measured in the follicular fluids using the method of Tappel and Zalkin (1959).

AST, ALT, AlP & AcP, Alkaline phosphatase (AIP; EC activity was measured at 405nm by the formation of paranitrophenol from para-nitrophenylophosphate as a substrate (Principato et al., 1985), however acid phosphatase (AcP; EC activity was measured by the method of Moss (1984) and Alanine aminotransferase (ALT; EC and asparatate aminotransferase (AST; EC activities were assayed by the method of Reitman and Frankel (1957).

Progesterone was determined by a simple solid phase ELISA Kit (Biochem Immunosystems, Italy) using horseradish peroxidase as a tracer.


Statistical analysis :

Data of biochemical components and progesterone were analysed by the 2-way analysis of variance (i.e. one way representing the physiological status of the ovary and the 2nd way representing the follicle size). (SAS, 1995). Also, data of ovaries according  to the different structures they bear are analysed by one way ANOVA. Mean comparisons were carried out by the Duncan Multiple Range Test (Steel and Torrie, 1990).



It has been found that 91% of the total ovarian samples contained at least one normal corpus luteum at either side. Whereas, 9% of the ovaries contained either cystic follicle or cystic corpus luteum or both structures.

As shown in Figure (1a) albumin concentration was highest in double ovulated (3.6g/dL), lowest in normal (2.1g/dL) with intermediate level in cystic (2.4g/dL) ovarian fluids. Similarly, the same trend was found with total protein concentration (Fig. 1b). Where, the protein level in double ovulated (403.3mg/ml) was found to be as much 1.7 times as in normal ovaries (233.9 mg/ml), however in cystic ovaries protein concentration (283.4mg/ml) was found to be as much 1.2 times as in normal fluids. Moreover, total protein levels increased as follicle size decreased (Table 1). However, the differences were not significant between various follicle size with respect to albumin levels. It has been found that protein and albumin content in follicular fluid was inversely related to follicular size (McNatty, 1978). Also, Albumin and protein concentrations in follicular fluid tended to be higher in atretic follicles. This may indicate that the likely estrogen and water uptake relationship in growing follicles may dilute follicular protein concentrations (Schuetz and Aniswicz, 1974). Double ovulation in cattle is undesirable trait because it reduces profitability and reproductive efficiency (Beerepoot et al., 1992). In this study all double ovulations were of dizygous pattern (i.e. two separate dominant follicles released two oocytes and resulted in two separate CLs). Apparently, the metabolic functions for double ovulations are yet unknown. Rather, the increased concentrations of albumin specifically and total protein in general might be ascribed to the higher need of peptides, enzymes and proteins for the growth and developmental processes required for producing two mature oocytes.


Table (1): Effect of follicle size of normal ovaries on progesterone and biochemical parameters in follicular fluids (Mean±SEM)


Follicle size

P  level




Albumin (g/dL)




> 0.05

Total protein (mg/ml)




< 0.01

Free radicals (nmol/ml)




> 0.10





< 0.01





< 0.01

Alk phos (U/L)




< 0.05

Acid phos (U/L)




< 0.01

Progesterone (ng/ml)




> 0.10

Means in the same row with different superscript are significantly different.


Free radicals concentration was not different (p>0.10) between small, medium and large follicles (Table 1), whereas the highest concentration was found in cystic follicles (Fig. 1c). This increase amounts 6 to 7 times more than normal or double ovulated FF. Chemistry of cystic fluids has not been fully elucidated, whereas such a high level of free radicles might be attributed to the hypoplasia of granulose cells inside such a follicle leading to some kind of abnormal reactions (Jainudeen and Hafez, 2000).

Alkaline and acid phosphatases((Fig. 2a & Fig. 2b, respectively)  were considerably (P<0.01) higher in double ovulated follicles than cystic or normal fluids. Similar results were found for AST (Fig. 2c) and ALT (Fig. 2d). Even though, the magnitude of increase in double follicles as compared to control was 7 and 40 times for alkaline and acid phosphatase, respectively. On the other hand this increase was 1.5 and 1.8 times for ALT and AST, respectively. Overall, the cystic follicles exhibited slightly higher enzyme concentrations than normal.

Within normal ovaries, small follicles contained significantly (P<0.05) higher enzyme activities than large follicles (Table 1). AST and ALT are important and critical enzymes in biological processes. They are involved in the breakdown of amino acids into -keto acid which are routed for complete metabolism through the Kreb’s cycle and electron transport chain (Harper et al., 1979). Moreover, alkaline phosphatase (AlP) was found to be a sensitive biomarker to metallic salts since it is a membrane bound enzyme related to the transport of various metabolites (Lakshm et al., 1991). The activity of AlP is concerned with energy metabolic activities in the body and the decrease in its activity may indicate impaired energy processing of the cells (Shakoori et al., 1994) Moreover, acid phosphatase (AcP) is a marker enzyme for lysosomes, thus related to general cell metabolism (Lakshmi et al., 1991). Increased follicular acid and alkaline phosphatase activity was found to be associated with small developing follicles than large ones (Stallcup, 1970). Furthermore, there are indications that normal (growing) follicles have less acid and alkaline phosphates activity than atretic follicles (Elfont et al., 1977). Though, the study of Lakshmi et al.  (1991) confirmed that alkaline and acid phosphatase activity decreased with follicular development (increased follicle diameter). The increase of such enzymes in double ovulated ovarian fluids might be a result of the higher metabolic activity required for growth and development of two oocytes.

Progesterone levels were not statistically different (P>0.10) between follicle size (Table 1), however, double ovulated or normal fluids contained higher (P<0.01) progesterone than cystic fluids (Fig. 1d). Most of the ovarian cysts in this study were of follicular cyst type, therefore less luteinization resulted in less P4 secretion. On the other hand, P4 concentration was higher in double follicles due to the higher luteinization process (higher lutein cell mass). Kesler et al. (1977) found that the concentrations of progesterone in periphoral plasma were normally low in cows with follicular cysts and higher in cows with luteal cysts and the later was attributed to the partial luteinization of the cysts.

In conclusion, ovary constitutes the primary sex organ in females, since it produces both the ova and sex hormones. As much attention has been paid to the extraovarian factors (i.e. gonadotrophin hormones) regulating folliculogenesis, follicle dominance, oocyte growth and development and ovulation. There are still much investigations need to be conducted as to closely monitor the intraovarian compounds controlling environment which surround the oocyte.

Moreover, this study indicates that, although the governmental regulations prohibit the slaughter of females less than 5 years old. There still are a high percent of the above 5 years old female buffaloes need to be subjected to more accurate check procedures (i.e. ultrasonography) before getting into slaughter.




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