MATERIALS AND METHODS

Sheep ovaries were collected from government approve slaughter house at Ziaguda, Hyderabad immediately after slaughter. The ovaries were washed with PBS and transported to the laboratory within 5-6 h after collection in a thermos flask containing warm water (37°C). On reaching the laboratory, working area was cleaned with 70% alcohol and the ovaries were handled aseptically. The oocytes were collected aseptically from the ovaries by slicing, aspiration and post aspiration slicing method. On aspiration the visible follicles present on the surface of the ovary were aspirated with 20 G needle fixed to 5 mL disposable syringe containing 2 mL of handling media. By slicing, the ovaries were held firmly with the help of forceps and were sliced into possible thin sections with a BP blade and washed down with a fine jet of handling media after slicing. The petriplate containing oocytes were placed for 5 min. For post aspiration slicing method the visible follicles present on the surface of the ovary were aspirated with 20 G needles fixed to 5 mL disposable syringe containing 2 mL of handling media. The aspirated ovaries were further subjected to slicing method in order to obtain the residual oocytes present in ovary. The oocytes were graded under a stereo zoom microscope and classified according to Rahman et al.¹⁵:

Only grades A and B oocytes were selected and subjected to in vitro maturation (IVM).

Experimental design: A series of experiments were designed and carried out to standardize the oocyte retrieval method, effect of season on sheep oocytes quality and quantity, effect of presence of corpus luteum on oocytes yield and optimum concentrations of leptin in culture medium to support growth and in vitro maturation of sheep oocytes:

Fig. 1:	Immature grade A oocyte

Fig. 2:	Immature grade B oocyte

The oocytes were washed 2 times with control medium separately. Only grade A and B oocytes were allowed to mature in 50 μL droplets of maturation medium in 35 mm petridishes. The droplets were overlaid with autoclaved and pre-equilibrated light weight mineral oil and kept for 24 h in a CO₂ incubator maintained at 39°C, 5% CO₂ and high humidity.

Fig. 3:	Immature grade C and D oocytes

A total 110 oocytes (Batches of 10 oocytes) were used, respectively in each group. In experiment I, the oocytes were subjected to in vitro maturation for 24 h in different concentration of leptin (0, 10, 20, 50 and 100 ng mL^–1) with TCM-199 where as for experiment II, TCM-199 supplemented with 10% FBS, 10 μg mL^–1 FSH, 10 IU mL^–1 LH, 1 μg mL^–1 and estradiol-17 β, 50 μg mL^–1. Gentamicin sulphate in different groups i.e., group I (Without leptin), group II (Leptin 10 ng mL^–1), group III (Leptin 20 ng mL^–1), group IV (Leptin 50 ng mL^–1) and group V (Leptin 100 ng mL^–1), respectably. The assessment of maturation was done by the degree of expansion of Cumulus-Oocyte Complexes (COC) and extrusion of first polar body (PB1) into perivitelline space (Fig. 4, 5).

Statistical analysis: Data on Cumulus-Oocyte Complexes (COC) expansion and extrusion of polar body were analyzed using software package used for statistical analysis (SPSS) (Version 17). One-way ANOVA between treatment groups. Differences among treatment groups were considered significant when p<0.05.

RESULTS AND DISCUSSION

A total 1520 sheep ovaries were procured from slaughter house. From those ovaries 3241 oocytes were retrieved by three methods like slicing, aspiration and post aspiration slicing method. A series of experiments were conducted to evaluate the efficacy of oocyte retrieval methods in relation to oocyte quality, effect of season on sheep oocytes quality and quantity, presence of corpus luteum on oocytes yield and compare the in vitro maturation (IVM) status of oocytes in different concentration of leptin 10, 20, 50 and 100 ng mL^–1 alone and in combination with traditional IVM medium.

Efficacy of oocyte retrieval methods in relation to oocyte quality: A total 3241 oocytes were retrieved by three methods like slicing, aspiration and post aspiration slicing method. The percentage of good quality oocytes was higher in the aspiration method (65.8%) as compared to the slicing (54.2%) and post aspiration slicing method (36.1%) (Table 1).

Fig. 4:	Cumulus-Oocyte Complexes (COC) expansion in matured oocyte quantity, presence of corpus luteum on oocytes yield and compare the in vitro maturation (IVM) status of oocytes in different concentration of leptin 10, 20, 50 and 100 ng mL–1 alone and in combination with traditional IVM medium

Evaluation of seasonal effect on oocytes recovery: The highest number of oocytes was recovered in the spring (March-April) season 1980 followed by winter (January-February) season 766 and in summer (May-June) season 495. Oocytes retrieved per ovary was highest in spring season 4.2±0.12 followed by winter season 3.35±0.14 and lower in summer season 3.1±0.10 (Table 2).

Effect of presence of Corpus Luteum (CL)on oocyte yield: The effect of presence of corpus luteum on oocyte yield was studied. Total 311 ovaries without CL and 104 ovaries with CL yields 586 oocytes and 143 oocytes, respectively. On an average 1.88±0.14 oocytes per ovary were recovered from ovaries without corpus luteum as compare to ovaries with corpus luteum 1.37±0.11 . The presence of corpus luteum on the ovary at the time of oocyte recovery significantly affected the quantity of oocytes (Table 3).

Effect of different concentration of leptin on in vitro maturation of sheep oocytes: The Cumulus-Oocyte Complexes (COC) expansion (73.80±1.59) was significantly higher in leptin 20 ng mL^–1 as compared with other concentrations 10 ng mL^–1 (67.74±1.94), 50 ng mL^–1 (44.47±2.59) and 100 ng mL^–1 (46.28±2.88) of leptin and polar body extrusion was significantly higher in leptin 20 ng mL^–1 (50.63±3.94) as compared with other concentrations leptin 10 ng mL^–1 (42.58±3.38), 50 ng mL^–1 (30.10±2.31) and leptin 100 ng mL^–1 (32.86±2.11) (Table 4).

Fig. 5:	First polar body extrusion into perivitelline space

Table 1:	Efficacy of oocyte retrieval methods in relation to oocyte quality
Different superscripts within a column for an attribute differ significantly by Duncan’s multiple range test (p<0.05)

Table 2:	Seasonal effect on oocytes recovery
Different superscripts within a column for an attribute differ significantly by Duncan’s multiple range test (p<0.05)

Table 3:	Effect of presence of corpus luteum on oocyte yield
Different superscripts within a column for an attribute differ significantly by Duncan’s multiple range test (p<0.05)

Table 4:	Effect of different concentration of leptin on in vitro maturation of sheep oocytes
Different superscript within the column are significantly different one-way ANOVA followed by Duncan’s multiple range test (p<0.05)

Table 5:	Effect of different concentration of leptin combination with IVM medium on in vitro maturation of sheep oocytes
Different superscript within the column are significantly different one way ANOVA followed by Duncan’s multiple range test (p<0.05)

Effect of different concentration of leptin in combination with IVM medium on in vitro maturation of sheep oocytes: The Cumulus-Oocyte Complexes (COC) expansion was highest in leptin 20 ng mL^–1 with IVM medium (81.90±1.87) when compared with other concentrations 10 ng mL^–1 (76.59±2.30), 50 ng mL^–1 (61.24±1.77) and 100 ng mL^–1 (58.58±2.86) of leptin with IVM medium. The oocytes cultured in leptin 20 ng mL^–1 (56.87±2.92) exhibited significantly higher percent of polar body extrusion, when compared with other concentrations 10 ng mL^–1 (40.99±3.11), 50 ng mL^–1 (33.97±2.19) and 100 ng mL^–1 (29.24 ±2.18) of leptin with IVM medium (Table 5).

DISCUSSION

Oocyte maturation is an essential physiological event for fertilization of oocytes in the present study, grade A and B quality oocytes recovery rate was higher in aspiration method (65.8%) followed by slicing method (54.25%) and post aspiration slicing (36.1%), the present study corroboration with the Majeed et al.¹². In present study, aspiration method was best it might be due to matured follicles were used for oocytes retrieval and less number of grade A and B quality oocytes recovered in slicing method may be due to isolation of oocytes from immature follicles. These reports also confirm the report of Pawshe et al.¹⁶. The grade A and B oocytes were retrieved greatly from aspiration technique and dissection technique when compared to grade C and D oocytes. However, the present results were not in accordance with Petrean et al.¹⁷ they observed that recovery of oocytes using the slicing technique in both ewes and lambs yielded more good quality oocytes per ovary than aspiration.

The present study advocated that there are significant differences between the three seasons that consequently affected the quality and the yield of oocytes that were comparable with the findings observed by Rust et al.¹⁴ with regard to number of oocytes recovered , oocyte recovery rate and oocyte quality. There was a tendency for the number of grade A oocytes to be higher during the cooler months and lower during the hotter months. The findings were in accordance with Zoheir et al.¹⁸ with regard to oocyte recovery and quality. In present study, quality and the yield of oocytes was low in summer season it may be due to the general tendency for a lower ovarian follicle density (Growth) during the warmer (Summer) months and after that a higher follicular population during the colder (Winter) months. Low follicular population during summer months might be due to day-light length is increased and poor nutrition.

The effect of presence of corpus luteum on oocyte yield was studied. In this study, luteal phase ovaries (With CL) yielded significantly lower numbers of oocytes 1.37±0.11 compared to non-luteal phase (Without CL) 1.88±0.14. The cause of a low number of visible follicles per ovary which were subjected to oocyte recovery by means of aspiration technique may be attributed to the fact that a large body of CL may inhibit the growth of follicles on the ovary and increase their atresia¹⁹. The present findings were in accordance with Davachi et al.²⁰ also observed that the presence of CL on ovaries lead to decrease in the quality and the quantity of oocytes.

However, the present results were not in accordance with Sahoo et al.²¹ they observed that the number of buffalo oocytes per ovary were 1.7±0.1 and 1.5±0.1 obtained from ovaries without CL and with CL, respectively.

In the present study Cumulus-Oocyte Complexes (COC) expansion (73.80±1.59) and meiotic resumption and development to M-II stage (50.63±3.94^a) exhibited significantly higher in leptin 20 ng mL^–1 as compared to 10, 50 and 100 ng mL^–1 of leptin. It may be due to leptin at this concentration was found to significantly increase stimulated progesterone, estradiol, production/secretion by cultured oocyte in a dose-dependent manner with higher concentrations of leptin inhibiting growth²². The present results were in accordance with Gabr et al.¹¹. They reported supplementation of leptin to maturation medium (TCM-199) at a level 20 ng mL^–1 increased in vitro nuclear maturation rate of oocytes, their fertilization rate and blastocyst production in camel oocytes. Similarly, Duggal et al.²³ reported with regard to high doses of leptin may have inhibitory effect on ovulations but did not influence steroid levels. However, present results were not in accordance with Craig et al.⁷ in pig, Boelhauve et al.²⁴, Paula-Lopes et al.²⁵ and Jia et al.²⁶ in bovine, Arias-Alvarez et al.⁹ in rabbit, Khaki et al.²⁷ in buffalo, when the antral oocytes were matured in the medium supplemented with leptin at concentration 10 ng mL^–1. This may be due to species variation, seasonal variation, concentration of leptin supplemented and stage of follicular development at the time of oocyte collection.

Effect of different concentration of leptin in combination with IVM medium on in vitro maturation of sheep oocytes. The Cumulus-Oocyte Complexes (COC) expansion was highest in leptin 20 ng mL^–1 (81.90±1.87) when compared with other concentrations 10 ng mL^–1 (76.59±2.30), 50 ng mL^–1 (61.24±1.77) and 100 ng mL^–1 (58.57±2.86) of leptin with IVM medium. This may be due to up-regulation of leptin receptors at 20 ng mL^–1. The oocytes cultured in leptin 20 ng mL^–1 (56.87±2.92) exhibited significantly higher percent of polar body extrusion when compared with other concentrations 10 ng mL^–1 (40.99±3.11), 50 ng mL^–1 (33.97±2.19) and 100 ng mL^–1 (29.24±2.18) of leptin with IVM medium. These observations were in accordance with Singh et al.²⁸ leptin at the concentration of 20 ng mL^–1 in maturation medium significantly (p<0.05) improved maturation rate, cleavage, morula and blastocyst development in buffaloes oocytes. In present study, leptin in combination with IVM medium showed best result, it may be due to the in vitro maturation (IVM) medium supplemented with 10% bovine serum albumin to prevent the zona pellucida hardening provides a source of albumin that balances the osmolarity and acts as a free radical scavenger. Whereas, gonadotropins and other growth factors helpful in achieving nuclear and cytoplasmic maturation it induce major alteration in the protein profile of oocytes²⁹.

However, present results were not in accordance with Cordova et al.³⁰ observed that oocytes matured in the presence of 1000 ng mL^–1 of leptin showed significantly higher percentage of tunel positive cells (6.9%) than matured in Fetal Calf Serum (FCS) (5.0%) and leptin receptor (LEPR) mRNA levels were significantly higher in the group matured with FCS. This may be due to variation in concentration of leptin supplemented and species.

CONCLUSION

It was concluded that on oocyte retrieval method percentage of grade A and B quality oocytes was higher in the aspiration method. On spring (March-April) season sheep oocytes quality and numbers was better as compare to winter and summer. The oocyte yield was significantly lower on ovary with corpus luteum. On in vitro development of sheep oocytes could be significantly improved through addition of leptin in culture media. Supplementation of leptin 20 ng mL^–1 with IVM shows best result as compare to leptin alone and control.

ACKNOWLEDGMENT

Author highly thankful to Department of Biotechnology (DBT), Government of India supported this study through a study grant to Dr. G. Aruna Kumari, Assistant Professor VGO, CVSc, Rajendra Nagar, 500030 Hyderabad, India.