By Gehan H. El-Sakkar*, Hamouda M. Ahmed** and Shahira … (59-76)… · · 2011-05-10siella...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 2 (April) 2008; 59– 76

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Histopathological, microbiological and biochemical studies on uteri and ovaries of infertile slaughtered buffaloes in Dakahlia Governorate

By Gehan H. El-Sakkar*, Hamouda M. Ahmed** and Shahira H.M.

Hussein*** *Pathology Dept., Zagazig Lab., Animal Health Research Institute

**Bacteriology Dept., Zagazig Lab., Animal Health Research Institute

SUMMARY

S eventy uterine and ovarian samples were collected from adult female buffaloes, slaughtered in different slaughter houses in Dakahlia Province. The animals

were suffering infertility. The specimens were examined bacteriologically, bio-chemically and pathologically. Ten blood samples (5 from normal and 5 from dis-eased buffaloes) were collected from the jugular vein, before slaughtering. The se-rum was separated and used for blood chemistry. The surface of uteri and ovaries were seared by a hot spatula and opened under complete aseptic precaution then a loopfull of the uterine content was directly cultured onto nutrient agar, MacConkey agar and blood agar. All the inoculated plates were incubated at 73°C for 24 h. The suspected colonies were examined culturally. Specimens were collected from the uteri and ovaries and fixed in 10% neutral buffered formalin. Five micron thick par-affin sections were prepared, stained by H & E and examined microscopically.

The most prevalent microorganisms were E. coli (10%), Klebsiella oxytoca (2.85%), Proteus mirabilis (2.85%), Staph. aureus (5.7%) and Strept. pyogens (7.1%). Uterine adhesion, endometritis and cystic ovaries were encountered. The uterus showed degenerative changes in the uterine glands with periglandular mono-nuclear cell infiltration and edema besides congestion and vasculitis. The examined ovaries showed either thick walled follicular cysts or leutein cysts with luteinized granulosa cells, hyperplastic theca-interna and theca-externa besides atretic follicles and hyperplastic lining of other follicles. The blood vessels were congested and showed thickened walls of the coiled arterioles. The estrogen was significantly in-creased, meanwhile the progesterone, T3 and t4 hormones were significantly de-creased. It could be concluded that the affection of the uterus and ovaries leads to infertility associated with hormonal disturbances. E. coli was the highest to isolate, but Kleb-siella oxytoca and Proteus merabilis were the lowest.

Referred byReferred by Prof. Dr. Othman A. Attallah Professor of Pathology, Fac. Vet. Med., Za-

gazig University Prof. Dr. Ahmed Ammar Professor of Microbiology, Fac. Vet. Med.,

Zagazig University


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INTRODUCTION

B uffalo is considered one of the most important multipurpose

farm animals. The infertility prob-lems are widely spread among this species causing excess reduction in their production. The ovarian le-sions appeared to be the main cause of infertility among buffaloes (Wahba et al 1995). The uterine disorders are contributing factors that impair the fertility of animals. The most common form of the uter-ine disorders is endometritis (Khan, 1994). This study was planned to iso-late the involved bacterial patho-gens describe the associated histo-pathological lesions and evaluate of the ovarian and thyroid hormones in buffaloes having cystic ovary to-gether with endometritis. Moreover, the antibiograms of some isolated pathogens were considered.

MATERIAL AND METHODS

S eventy uterine and ovarian samples were collected from

slaughtered adult female buffaloes with a history of infertility the sam-ples were collected from Mit-Ghamr, Kom-El Nour, Mit-Yaeesh and Atmeda abattoirs during the pe-riod from August, 2005 till Novem-ber 2005 in Dakahlia Governorate. The surface of uterus and ova-ries were seared by a hot spatula

and opened under complete aseptic precaution then a loopfull of the uterine content was directly cul-tured onto nutrient agar, MacCon-key agar and blood agar. All the in-oculated plates were incubated at 37°C for 24 h. The suspected colo-nies were examined culturally bio-chemically as described by Carter (1984). Drug sensitivity test was done for all the bacterial isolates according to Cruickshank et al (1975). Ten blood samples were col-lected from the jugular vein (before slaughtering) of 5 buffaloes suffer-ing from cystic ovary with endo-metritis and other 5 healthy buffa-loes. Serum was obtained by cen-trifugation at 3000 rpm for 20 min, stored at –20°C and used for hor-monal assay. Serum progesterone was estimated by Radioimmunoas-say (RIA) according to Kubasik et al (1984), estrogen according to Xing et al (1983) and thyroxin (T4 ) together with triiodothyronine (T3) according to Abraham (1981). Specimens were collected from the uterus and ovaries and im-mediately fixed in 10% neutral buffered formalin. Five micron thick paraffin sections were pre-pared and stained with hematoxylin and eosin (Bancroft and Gamble, 2002) and examined microscopi-cally.


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Statistical Analysis: The obtained results were sta-

tistically analyzed using Student t-test as described by Petrie and Watson (1999).

RESULTS AND DISCUSSION

1-Microbiological

T he uterine and ovarian sam-ples, from 70 slaughtered buf-

faloes, yielded 20 isolates (28.5%) as shown in table (1). The most prevalent organisms were E. coli (10%), Klebsiella oxytoca (2.85%),

Proteus mirabi l i s (2 .85%), Staph.aureus (5.7%) and Strept. pyogenes (7.1%). These findings are in partial agreement with those obtained by El-Sawaf et al (1961) and Fadel (2000) who recorded isolation rates of 25% and 20.60% respectively. These results are not in concurrence with Shouman et al (1977), Son-Changho et al (1998) and Barakat et al (2005) who recorded isolation rates of 7%, 15.68% and 14% respectively. Table (2) shows the results of

Number of samples

Total isolates and percentages

Total E. coli Klebsiella oxytoca

Proteus mirabilis

Staph. aureus

Strep. pyogen

No. % No. % No. % No. % No. % No. %

70 sample 20 28.5 7 10 2 2.85 2 2.85 4 5.7 5 7.1

Table (1): The bacterial isolates from the 70 examined samples.

Tested antibiotic Disc con

E. coli (7)

Kl. oxy (2)

Pr. m (2)

Staph. aureus

(4) St. py

(5) Total (20)

No. % No. % No. % No. % No. % No. % Amoxycillin 30 4 57.1 1 50 1 50 3 75 4 80 13 65 Enrofloxacin 5 5 71.4 2 100 1 50 3 75 3 60 14 70 Erythromicin 15 1 14.3 0 0 0 0 2 50 4 80 7 35 Florofincol 30 3 42.9 1 50 2 100 4 100 5 100 15 75 Gentamycin 10 6 85.7 2 100 1 50 2 50 2 40 13 65

Oxytetrcyclin 30 1 14.3 1 50 0 0 2 50 2 40 6 30

Table (2): Sensitivity test of the bacterial isolate.


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pyogens. The total isolated micro-organisms were highly sensitive to florafincol (75%), Enrofloxacin (70%), amoxycillin (65%), genta-mycin (65%), erythromycin (35%) and oxytetracycline (30%). These results are in a partial agreement with Awad and El-Hariri (1980)and Pradhan et al (1999). They reported that enrofloxacin, genta-mycin and chloramphenicol were highly effective against the isolates from bovine endometritis. On the other hand the obtained results dis-agree with Hussein et al (1993) who stated that tetracycline and ampicillin are the most effective in treating repeat breeder syndrome. Hormonal:

Table (3) indicates that the estrogen level was significantly high meanwhile the progesterone and both the triiodothyronine (T3) and thyroxin (T4) were signifi-cantly low in the buffaloes suffer-ing both cystic ovary and endo-metritis. These results agree with Cristofori et al (1986), Saleh et al (2000) and Mohamed and Hus-sein (2004) in the camel and buffa-loes. Douthwaite and Dobson (2000) mentioned that the serum progesterone level was lower in cows with follicular cyst than in cows with leutein cysts. These re-sults may be attributed to less lu-

teinization of the follicular cyst with low progesterone secretion (Zeitoun, 2003). The estrogen level in this study was high in buf-faloes suffering from cystic ovary with endometritis. This goes along with the results of Mabrouk (1989) and Homeida et al (1991) who found a close correlation be-tween the follicle size and estrogen level. Homeida et al (1988) ob-served that the estrogen concentra-tion was parallel to follicle size. They suggested that the follicle se-cretes estrogen. Similar results were reported by Hamilton et al (1995) who observed a higher con-centration of estrogen in cow with cystic ovary than those with ovula-tory cycles. The current work showed that the levels of the T4 and T3 were low in the serum of buffaloes suffering from cystic ovary with endometritis. These re-sults are in agreement with those of Metwelly et al (2004) who re-ported that the levels of both the T4 and T3 were the lowest in cases of cystic ovary associated with endo-metritis in she camel. Abdo et al (1969) reported that the low thy-roid function plays a role in the oc-currence of cystic ovaries.


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Macroscopically, some ex-amined uteri showed endometritis and others showed uterine adhe-sions. The inflamed endometrium was swollen and covered with tur-bid exudate. Some cases showed grayish endometrium with rough surface and irregularly dark red patches. Similar lesions were de-scribed by Hegazy et al. (1998) and Moustafa et al. (2002).

Almost, all the examined ov-aries were cystic, in one or both ovaries. Unilateral or bilateral hemorrhagic cysts were found (Fig. 1). Similar findings were pre-viously noticed by Selim et al. (1998) in the right ovary only. Some ovaries revealed an-encaps-ulated, white and firm mass (fibr-oma). Jones et al. (1997) reported

that fibroma was the more com-mon ovarian neoplasms in cows. Histopathological examination of uteri: Microscopically, the endo-metrium of the affected buffaloes showed mononuclear cell infiltra-tion (Fig. 2), in addition to severe congestion in the lamina propria (Fig. 3). Similar results were de-scribed by El-Mashad (1998), and Refat and Badr (2006). Variable degenerative changes were found in the uterine glands (Fig. 4). Al-most similar results were reported by Hegazy et al. (1979), Amer et al. (2001) and Kubar and Jalakas (2002). Moreover, periglandular mononuclear cell infiltration and edema were found (Fig. 5). Similar

Table (3): Some blood hormones in healthy buffaloes and in those suffer-ing from cystic ovary and endometritis (N=5).

Ovarian finding Number of

buffaloes

Proges-terone

(mg/ml)

Estrogen (Pg/ml)

T3 (ng/ml)

T4 (ng/ml)

T3 : T4 (ratio)

Healthy ovary and uterus

5 1.98 ± 0.13

62.72 ± 6.31

2.36 ± 0.11

7.13 ± 0.05

0.33 ± 0.03

Cystic ovary and endometritis

5 1.31 ± 0.22*

81.42 ± 3.51*

1.85 ±0.19*

4.26 ±1.13*

0.42 ± 0.02*

* Significant at P < 0.05


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results were described by Hegazy et al. (1979 & 1998) and El-Mashad (1998). The basilar uter-ine glands and adjacent stroma in-vaded the myometrium (adenom-yosis) along the blood vessels (Fig. 6). Such lesion is triggered by the excessive estrogen production by the cystic follicles (Jones et al., 1997). The elevated estrogen level led to estrogen – mediated endo-metrial hyperplasia. The deep layer of the lamina showed thickened walls of the spiral arterioles, be-sides edema (Fig. 7). Similar re-sults were described by Mahdy (1988) and El-Mashad (1998). The variation in intensity of the uterine inflammatory changes was attributed to the host resistance, en-vironment, virulence of microor-ganisms and hormonal effect Amer (1998) and Abd El-Wahab (1991). Histopathological examination of ovaries: Microscopically, the exam-ined affected ovaries showed fol-licular cyst with thick walls (Figs. 8 & 9). Similar findings were pre-viously described (Al-Dahash and David, 1977; Hussein and Has-sieb, 1989; Selim et al., 1998 and Amer et al., 2001). Other cases showed lutein cysts with hyper-plastic theca interna and externa

(Fig. 10). They developed from the most mature follicles due to dis-turbed secretion and release of the gonadotropin, beisdes hypothy-roidism and L.H deficiency (Abdo et al., 1969 and Omar et al., 1982). Atretic follicles and degen-erated lutein cysts were detected (Figs. 11 & 12). Similar results were described by Hussein and Hassieb (1989) and Wahba (1998). An ovary of aged buffalo showed fibroma consisting of in-terlacing bundles of fibrous con-nective tissue without any malig-nancy (Fig. 13). Other case showed fibroma with spindle shaped nuclei arranged in wavy bundles (Fig. 14). Similar tumor in uterus was described by Abd El-Mottaleb and Badr (2007) .

It could be concluded that the endometrial lesions are among the most common causes of infertility. Moreover, the ovarian damage contributed to the problem.


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Fig. (2): Uterus of buffalo-cow showing mononuclear cell infiltration

(arrows) in the inner lamina propria (H & E., x 100).

Fig. (1): Ovaries of aged buffalo-cow showing hemorrhagic follicles and

cysts (arrows).


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Fig. (4): Uterus of buffalo-cow showing variable degenerative changes in the uterine glands. (H & E., x 400).

Fig. (3): Uterus of buffalo-cow showing severe congestion in the lamina

propria (C)


67 Fig. (6): Uterus of buffalo-cow showing adenomyosis (arrows) (H & E.,

x 100).

Fig. (5): Uterus of buffalo-cow showing periglandular mononuclear cell infiltration (m) and edema (arrows) (H & E., x 100).


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Fig. (8): Ovary of aged buffalo-cow showing follicular cyst (arrow) with thickened wall (H & E x 4).

Fig. (7): Uterus of buffalo-cow showing thickened walls of spiral arteri-oles (v) in the depth of lamina propria besides edema (arrows) (H & E., x 100).


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Fig. (10): Ovary of aged buffalo-cow showing lutein cyst with hyperplas-tic theca interna (I) and theca externa (E) (H & E., x 100).

Fig. (9): A high power of Fig. (8) to show the thickened wall of a follicular cyst (H & E., x 200).


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Fig. (12): Ovary of buffalo-cow showing degenerated lutein cyst (H & E., x 200).

Fig. (11): Ovary of aged buffalo-cow showing atretic follicles (H & E, x 100).


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Fig. (14): Ovary of aged buffalo-cow showing fibroma with spindle shaped nuclei arranged in wavy bundles (H & E., x 200).

Fig. (13): Ovary of aged buffalo-cow showing fibroma Consisting of inter-lacing bundles of fibrous connective tissue (H & E., x 100).


72

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