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Histoenzymic Studies on the Ovary of Indian Buffalo During Different Reproductive Stages

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Histoenzymic Studies on the Ovary of Indian Buffalo During Different Reproductive Stages Original Article Buffalo Bulletin (January-March 2019) Vol.38 No.1 HISTOENZYMIC STUDIES ON THE OVARY OF INDIAN BUFFALO DURING DIFFERENT REPRODUCTIVE STAGES Devendra Pathak* and Neelam Bansal ABSTRACT cells of secondary and tertiary follicles while it was moderate in the theca cells. Most of the Distribution pattern of phosphatases, enzymes were more active in pubertal and pregnant oxidoreductases and non-specifc esterases were buffaloes compared with prepubertal buffaloes and studied in ovary of prepubertal, follicular phase, could be correlated with the steroid synthesis. The luteal phase and in pregnant animals. In the ovary of difference in the intensity of enzyme in different prepubertal buffaloes, distribution of phosphatases compartments of ovary also correlated with the (AKPase and G-6-Pase) was negligible in the cells participating in the hormone synthesis. surface epithelium, tunica albuginea and stroma in the cortex region while it was weak during the Keywords: Bubalus bubalis, buffalo, follicular, luteal and pregnant animals. Its activity histoenzymic study, ovary, reproductive stages was strong in the primordial and primary follicles in all the phases of the reproduction. Strong to intense reaction was observed in the theca cells and INTRODUCTION granulosa cells. The luteal cells and blood vessels in corpus luteum of luteal phase showed moderate Buffalo is one of the important livestock reactions while strong reaction was observed in resources of India and it plays a vital role in the pregnant buffaloes. The enzyme reaction of improving the socio-economic status of rural dehydrogenases revealed that the steroidogenic masses. Buffalo can be considered as a pillar for cells were more active during follicular phase, the development of dairy industry in India as the luteal phase and pregnancy while weak activity species is a major contributor in country’s milk was observed in the surface epithelium and tunica production (56.00% of total milk), despite the fact albuginea. Reactivity of diaphorases were weak that they constitute only 34.60% of total bovine to moderate in surface epithelium and tunica population, and this can be attributed to steady albuginea, moderate activity in theca cells and increase in buffalo population in India during last oocyte and intense reaction in granulosa cells of two decades, which is about 1.93% (FAO, 2012). tertiary follicles and weak activity in connective Delayed puberty, poor expression of estrus, high tissue septa and intense in luteal cells in parenchyma incidence of early embryonic mortality, long inter- of corpus luteum of buffalo. Activity of NSE was calving period and seasonal variation in fertility strong in the granulosa cells and corona radiate compromise the productivity of buffalo (Bubalus Department of Veterinary Anatomy, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India, *E-mail: [email protected] 19 Buffalo Bulletin (January-March 2019) Vol.38 No.1 bubalis). A coordinated relationship exists between with different substrates for the demonstration hypothalamus, pituitary and ovary of animals and of phosphatases, oxidoreductases, esterases as ovary undergoes changes in its activity of cell described in Table 1. The positive and negative types based on infuences from hypothalamus and controls were carried out wherever possible. pituitary at different stages of reproduction. The location and the reactivity of various enzymes vary during different stages of reproduction and they RESULTS AND DISCUSSION play important role at each stage of reproductive physiology. The complete study of various enzymes Distribution of phosphatases, during different stages of reproduction is required oxidoreductases and non-specifc esterases were to understand the follicular development and studied in ovary of prepubertal (Table 2), follicular steroidogenic activity of ovaries in buffaloes. Thus phase (Table 3), luteal phase (Table 4) and in the present investigation was designed to elucidate pregnant animals (Table. 5). phosphatases, oxidoreductases and non-specifc esterases in ovary during prepubertal, follicular Phosphatases phase, luteal phase and pregnant buffaloes. Alkaline phosphatases (AKPase) In the ovary of prepubertal buffaloes, distribution of AKPase was negligible in the MATERIALS AND METHODS surface epithelium, tunica albuginea and stroma in the cortex region while it was weak during the The ovaries of buffaloes (n = 52) were follicular, luteal and pregnant animals. Its activity collected from New Delhi Municipal Corporation was strong in the primordial and primary follicles Abattoir, Gajipur; M.K. Overseas Pvt. Ltd., Dera in all the phases of the reproduction (Figure 1(a)). Basi, local abattoir at Bareilly and Teaching AKPase is known for synthesis and transport of Veterinary Clinical Complex, GADVASU, substances across the cell membrane and thus it Ludhiana. They were classifed into four groups, might be required for growth and differentiation of prepubertal (based on the history and age of ovarian follicles. Roy and Uppal (2005) discussed the animal), follicular phase (presence of a that this enzymes occurred in cells specialized dominant follicle on the surface and absence of for endocytosis and pinocytosis. This enzyme corpus luteum), Luteal phase (presence of a fully has been found to be involved in energy transfer developed corpus luteum), pregnant (presence of reactions (Freeland and Szepesi 1971). Strong to foetus in the uterus). intense reaction was observed in the theca cells and Fresh unfxed tissues from ovary of Indian granulosa cells (Figure 1(b)). Weak reaction was buffaloes were collected from slaughter houses observed in the oocyte and zona pellucida while immediately after the slaughter of the animals intense reactions were noted in the corona radiate and placed in tissue freezing medium (Leica) and cells in all the stages of reproductive cycle (Figure frozen in liquid nitrogen. Cryostat sections of 6 1(c)). The distribution of this enzyme was moderate to 7 µm thicknesses at -20°C were obtained on in the ovarian medulla during prepubertal period glass slides with cryostat microtome and incubated while weak to moderate during all pubertal stages. 20 Buffalo Bulletin (January-March 2019) Vol.38 No.1 Table 1. Histoenzymic techniques used on cryostat sections of ovary. Incubation Sr. No Enzyme Substrate Method Reference Time (minute) A. Phosphatases Naphthol AS-MX phosphate Simultaneous coupling azo Alkaline phosphatise Barka and i. disodium salt in combination dye method using substituted 30 (AKPase) Anderson (1963) with Fast Blue RR naphthol Glucose-6- phosphatase Glucose-6-phosphate and Lead nitrate method of Barka and ii. 20 (G-6-Pase) lead nitrate Wachstein and Meisel (1956) Anderson (1963) B. Oxidoreductases Succinic Dehydrogenase Standard method of Bound iii. Di-NA-succinate 15 Pearse (1972) (SDH) enzyme by Nitro BT method Lactate dehydrogenase Standard method of Bound iv. Na-DL-lactate 30 Pearse (1972) (LDH) enzyme by Nitro BT method 21 Glutamic dehydrogenase Standard method of bound v. Na-L-glutamate 15 Pearse (1972) (GLD) enzyme by Nitro BT method Glucose-6-phosphate Standard method of bound vi. Di-Na-Glucose-6-Phosphate 30 Pearse (1972) dehydrogenase (G-6-PD) enzyme by Nitro BT method Reduced nicotinamide adenine dinucleotide Standard method of Bound vii. Co-enzyme (NADPH) 35 Pearse (1972) phosphate diaphorase enzyme by Nitro BT method (NADPH-diaphorase) Reduced nicotinamide adenine dinucleotide Standard method of Bound vi. Co-enzyme (NADH) 35 Pearse (1972) diaphorase (NADH- enzyme by Nitro BT method diaphorase) Monoamine oxidase Standard method of Bound vii. Tryptamine hydrochloride 60 Pearse (1972) (MAO) enzyme by Nitro BT method Buffalo Bulletin (January-March 2019) Vol.38 No.1 Table 1. Histoenzymic techniques used on cryostat sections of ovary. (Continue) Incubation Sr. No Enzyme Substrate Method Reference Time (minute) A. Phosphatases C. Esterases Non-specifc esterase Barka and viii. Alpha naphthol Acetate Naphthol acetate method 10 (NSE) Anderson (1963) Table 2. Enzyme histochemistry on the ovary of prepubertal buffalo. Cortex Enzyme/Ovarian Follicle Medulla components Secondary Graafan/Tertian 22 SE TA CS P TH MG ZP O TH MG ZP O IGC MS BV RO HG AKPase OOO +++ +++/+++ ++ - + +++/++++ ++ - + ++/+++ O +++ +/++ - G-6-Pase + + + +/++ + + - - + + - - + + + + - G-6-PD O/+ O/+ O/+ + +/++ + - - +/++ + - - + O/+ + + - SDH + + O/+ + O/+ O/+ -- O/+ O/+ - - + O/+ + + - GLD + + O/+ + + + - - + + - - ++ O/+ + + - LDH ++ + + ++ + ++ - - O/+ +/++ - - +/++ + + O/+ - NADH diaphorase + O/+ + ++++ ++++ +++ O + +++/++++ ++ O + ++ + + ++ - NADPH diaphorase + O/+ O/+ +++ +++ + O + +++ + O + ++ O/+ + + - MAO O/+ O/+ O/+ ++ + + - - + + - - + O/+ O/+ + - NSE + + O/+ + O/+ + - - O/+ + - - + O/+ + + - - Not Observed, O-Absent; + Weak; ++ Moderate; +++ Strong, ++++ Intense SE- Surface epithelium, TA-Tunica albuginea, CS -cortical Stroma, P-Primordial and Primary follicle TH- theca, MG-Membrana Granulosa, ZP- Zona Plellucida, O-Oocyte IG-Interstial gland, MS- Medullary Stroma, HG-Hilar Gland, BV- Blood vessel Buffalo Bulletin (January-March 2019) Vol.38 No.1 Table 3. Enzyme histochemistry on the ovary of buffalo during follicular phase. Cortex Enzyme/ Follicle Medulla Corpus Ovarian Secondary Graafan/Tertian Luteum components SE TA CS P TH MG ZP O TH MG ZP O IG MS BV HG AKPase O O O/+ +++ +++/++++ ++/+++ O + +++/++++ ++/+++ - + ++ O/+ +++ + ++ G-6-Pse + + + ++ ++ + - - ++ + - - ++ + + + ++ G-6-PD O/+ O/+ O/+ +++ +++
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