The present study was conducted to assess the comparative efficacy of three oocyte harvesting techniques viz., aspiration, puncture and slicing methods on oocyte recovery in goat ovaries obtained from municipal slaughter house, Jammu. Among the three collection methods, the slicing technique yielded the highest number of total and good quality oocytes (7.03±0.21 and 2.84±0.16, respectively) followed by the puncture (3.84±0.26 and 1.03±0.12, respectively) and the aspiration methods (2.65±0.2 and 0.61±0.13, respectively). Ovaries without CL yielded significantly higher (P≤ 0.05) number (5.30±0.20) of oocytes than CL bearing ovaries (3.18±0.19). Corpus luteum bearing ovaries had also lower follicular population than ovaries without CL. It was concluded from the present study that slicing method is superior to the other two methods employed in this study to harvest a greater number of good quality oocytes from goat ovaries. Corpus luteum has negative effect on follicular population and oocyte yield from these ovaries.
The development and application of assisted reproductive technologies like In vitro production of embryos (IVP) through In vitro maturation (IVM), fertilization and culture of oocyte can revolutionize the research in domestic livestock. For any successful IVP program artificial removal of cumulus-oocyte complexes (COCs) from follicles, culturing, and maturing them is a primary requirement where the optimal rates of embryo production In vitro can be attained by selecting ovaries and follicles, which provide oocytes to undergo maturation, successful fertilization, and In vitro development (Rahman et al., 2008). Extensive research on In vitromaturation (IVM), In vitro fertilization (IVF) and in vitro culture (IVC) of the resulting zygotes in sheep and goat has been reported by Cognie et al. (2003), but limited information has been reported on the evaluation of goat ovaries or methods for the efficient collection and grading of oocytes. The number of high quality oocytes harvested from an ovary is an important aspect in the In vitro maturation and production of embryos. Oocytes for various assisted reproductive technologies are collected from different sources like oviducts soon after ovulation, mature follicles shortly before ovulation or immature and atretic follicles usually from abattoir material. The raw material generally used for the production of domestic animal embryos are ovaries of abattoir origin and these ovaries are the cheapest and most abundant source of primary oocytes for large scale production of mature oocytes through In vitro maturation (Agarwal et al., 1995). The present work was therefore undertaken with the objective of evaluating the efficacy of three different methods of oocyte recovery viz. aspiration, puncture and slicing along with the effect of presence/absence of corpus luteum (CL) and follicular development on oocyte recovery from abattoir derived goat ovaries.
Materials and Methods
Seventy eight ovaries of goats aged between 2-4 years with unknown reproductive histories were obtained from municipal slaughter house, Jammu and transported to the laboratory in a thermos containing Dulbecco’s phosphate buffer saline (DBPS) at 37°C within an hour of slaughter. In the laboratory each ovary was separated from the surrounding tissue and overlying bursa. The ovaries were rinsed in physiological saline and 70% alcohol followed by three washings in DBPS with antibiotics (Penicillin and Streptomycin). The presence or absence of corpus luteum was recorded. The surface follicles visible to the naked eye were classified into three groups depending on their diameter: small (<2 mm), medium (2-4 mm) and large (>4 mm) (Shailaja and Kumari, 1984) and recorded accordingly. The ovaries were then transferred to 90 mm petri dishes containing holding medium (HEPES supplemented TCM 199, Sigma Aldrich, USA and Gentamycin sulphate 50 µg/ml). Each ovary was processed individually and oocytes were collected by one of the following methods-
In all three methods the petri dishes were kept undisturbed for 5 minutes to allow COCs to settle. The Petri dishes were then examined under stereo zoom microscope and the oocytes were scanned. The scanned oocytes were separated into a 35 mm petri dish for grading at 63x magnification and then assessed as good, fair and poor based on cumulus investments, their compactness and ooplasm granulity (Das et al., 1996). The number and quality of oocytes were recorded for each ovary. Statistical analysis to compare the efficacy of different collection methods was performed by one way anova and effect of presence of corpus luteum and follicular population was analyzed by student’s t-test (Snedecor and Cochran 1989).
Results and Discussion
The mean oocyte recovery in the present study was 4.51±0.25, of which 1.50±0.13 (33.24%), 1.76±0.13 (39.20%) and 1.26±0.74 (27.56%) were of good, fair and poor grade oocytes, respectively (Table 1). The mean oocyte recovery rate recorded in this study was similar to the earlier findings of Pawshe et al. (1994) and Das et al. (loc cit) in goats and buffaloes, respectively. However Wani et al. (1999) in sheep and Wang et al. (2007) in goat have reported higher mean recovery rate in comparison to the present study.
Table 1: Effect of harvesting technique on the quantity and quality of oocytes recovered from goat ovaries (Mean ± S.E)
|Harvesting technique||No. of ovaries||No. of oocytes recovered||Oocyte quality|
|Overall||78||352||1.50±0.13 (33.24%)||1.76±0.13 (39.20%)||1.26±0.74 (27.56%)||4.51±0.25|
Means with different superscripts within a column vary significantly (P<0.01); Figures in parentheses indicate percentage.
Among the three harvesting techniques, the slicing method appeared to be superior in terms of both total recovery and number of good and fair grade oocytes. This method yielded a significantly (P<0.01) higher number of oocytes (7.03±0.21) per ovary compared to the puncture (3.84±0.26) and aspiration methods (2.65±0.23). Similar findings have been reported Pawshe et al. (loc cit) in goats and Das et al. (loc cit) in buffaloes. In contrast Wani et al. (2000) did not find any advantage of using slicing over puncture technique as both techniques yielded almost similar number of oocytes. Aspiration seems to be least efficient in terms of both total recovery and yield of good and fair grade oocytes, however Hoque et al. (2011) reported that the number of normal COCs per ovary is significantly higher (p < 0.01) in aspiration (2.48) followed by slicing (1.91) and puncture (1.85) techniques. Higher oocyte recovery in ovarian slicing may be due to their release from both surface follicles as well as from deeper cortex (Das et al., loc cit). It may be difficult to aspirate oocytes from small and medium sized follicles before cumulus expansion (Ball et al., 1983), however, the extra pressure applied during puncture may release oocytes from these follicles. The results clearly indicate that collection of oocytes by the slicing technique produces a greater number of total as well as good and fair (usable) grade oocytes.
The mean number of total (7.49±0.27), medium (1.78±0.10) and large follicles (1.05±0.09) in 51 ovaries without CL were significantly higher (P≤ 0.05) than in 27 CL bearing ovaries (5.55±0.22, 0.77±0.08 and 0.44±0.09, respectively (Table 2), however the number of small sized follicles was almost similar in both types of ovaries. The presence of CL decreases follicular activity, which has also been reported in sheep (Alsafy and El-Shahat, 2011) and cattle (Pierson and Ginther, 1987). Inhibiting effects of the CL on follicular growth may be due to the secretion of progesterone which prevents the release of FSH from anterior pituitary.
Table 2: Effect of presence of corpus luteum and follicular population on oocyte recovery from goat ovaries (Mean ± S.E)
|Ovary Characteristics||No. of Ovaries||No. of Follicles||Oocytes Recovered|
Means with different superscripts within a column vary significantly (P<0.05)
The mean number of oocytes from ovaries without CL (5.30±0.20) was significantly higher (P≤ 0.05) than from CL bearing ovaries (3.18±0.19). Similar results were also reported in buffaloes (Das et al., loc cit) and sheep (Alsafy and El-Shahat, loc cit). The presence of CL decreases the oocyte yield per ovary which might be due to the major portion of ovary being occupied by lutein cells.
The first author is thankful to University Grants Commission for providing financial assistance under Maulana Azad National Fellowship for minority students during the study.