Experimental site:
The experiment was conducted in the laboratory of the Dept. of Animal Production & Management and Dept. of Animal Nutrition, Genetics & Breeding, Faculty of Animal Science & Veterinary Medicine, Sher-e-Bangla Agricultural University.
Collection of ovaries and processing:
Collection of ovaries and processing have been performed according to the slight modification as described previously (Jamil et al., 2008). Briefly, ovaries from sexually mature goats were collected within 30 min of slaughter from the slaughterhouse. During collection, ovary was marked whether it is right or left and the presence or absence of corpus luteum (CL) was also recorded. They were then transported within 2 h of slaughter to the laboratory in a vacuum flask containing sterilized phosphate-buffered saline (PBS) at 25-30°C. Ovary collection times were classified into 2 breeding seasons, i.e. Summer season (July, August and September) and Winter season (December, January and February). Upon arrival at the laboratory, the ovaries were then transferred to sterilized petridishes and rinsed thoroughly by phosphate buffer saline at 25°C before further processing. The adipose tissues and surrounding bursa were removed from ovaries surface.
Evaluation of ovary:
After collection and trimming ovaries were weighed in gm in a digital balance and the weight was recorded in tabular form. The length and width in cm of left, right, CL-present and CL-absent ovaries measured with digital slide calipers. There were the different size of follicles on the surface of both ovaries. The number of visible follicles on the surface (with or without CL) of ovaries were counted and recorded for further analysis.
Oocyte collection and COCs evaluation:
The ovaries were washed 2-3 times in phosphate buffer solution (PBS) at 30°C. They were then placed in a beaker and kept in a water bath at 30°C. Each ovary was individually handled, and oocytes were recovered by the following way according to Ramsingh et al. (2013). In the aspiration technique, 18-gauge hypodermic needles were used to puncture the ovarian surface. The 10 ml syringe was loaded with D-PBS (1.0-1.5ml) and the needle (18G) was put in the ovary parenchyma near the vesicular follicles and all follicles were aspirated near the point at the same time. After aspirating the follicles from one ovary, the aspirated follicular materials were transferred slowly into 35-mm Petridishes, avoiding damage to the cumulus cells. Then the petridishes were kept undisturbed for 5 minutes, allowing the COCs to settle down. Then the COCs were searched and graded under an inverted microscope (Olympus, Tokyo, Japan) at low magnification.
Categories of COCs:
The COCs were classified according to the method of Khandoker et al. (2001) with slight modification. Briefly, quality of cumulus-oocyte complexes were analyzed based on layers of granulose cells. There are four categories of cumulus-oocyte complexes: category A (good quality), oocytes surrounded by cumulus cells with large quantities (more than 3 layers) and compact with a homogeneous ooplasm, category B (medium quality) the oocytes partially surrounded by less than 3 layers of cumulus cells with homogeneous ooplasm, category C (poor quality) the oocytes not covered by cumulus cells or oocytes surrounded by cumulus cells slightly and category D: degeneration observed both in oocytes and cumulus cells. Here grade A and B were considered as normal COCs and grade C and D as abnormal COCs.
Statistical analysis:
All values were expressed as Mean±SE (Standard Error). The p-value greater than 0.05 were considered as non-significant and the p-value less than 0.05 were considered as significant. Data were analyzed with the SAS (Statistical Analysis System) software using one-way Analysis of Variance (ANOVA).