Oocyte Retrieval Methods, Grade and Percentage of Oocytes in Goats  

L. Ram Singh1 , K. Sadasiva Rao2 , K. Murali Mohan3
1. Assistant Professor, Department of Animal Reproduction, Gyanecology and Obstetrics
2. Professor & Univ. Head, Dept. of VGO, CVSc, Rajendranagar, Hyderabad- 500 030 (A.P.) INDIA
3. Assistant Professor, Dept. of VGO, CVSc, Rajendranagar, Hyderabad- 500 030 (A.P.) INDIA
Author    Correspondence author
International Journal of Molecular Veterinary Research, 2013, Vol. 3, No. 2   doi: 10.5376/ijmvr.2013.03.0002
Received: 15 Feb., 2013    Accepted: 04 Mar., 2013    Published: 21 Mar., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Singh et al., 2013, Oocyte Retrieval Methods, Grade and Percentage of Oocytes in Goats, International Journal of Molecular Veterinary Research, Vol.3, No.2 4-6 (doi: 10.5376/ ijmvr.2013.03.0002)

Abstract

The present study was undertaken to assess the relative efficiency of three different collection techniques of percentage and grades of oocytes in goats of Hyderabad. The mean oocyte recovery from aspiration technique, 2.15 ± 0.31 and 2.9 ± 0.46 in dissection technique and 6.55 ± 0.57 and 4.51 ± 0.46 in slicing technique. The mean oocyte recovery rate was 1.5 ± 0.22 and 1.0 ± 0.33 under aspiration technique, 2.58 ± 0.47 and 2.43 ± 0.36 under dissection technique and 5.26 ± 0.46 and 6.3 ± 0.71. The mean numbers of oocytes of different grades recovered were 0.34 ± 0.03, 0.62 ± 0.07 and 1.37 ± 0.09 in aspiration, dissection and slicing techniques, respectively. Among the different grades of oocytes, significantly higher per cent of A (27.78) and B (38.89) grade oocytes were retrieved by aspiration and dissection technique when compared to slicing technique. With respect to C and D grade oocytes, significantly higher numbers of oocytes were retrieved by slicing technique than the aspiration and dissection techniques. Out of 437 oocytes retrieved in the present study, significantly higher per cent (50.57) yield was by slicing technique, followed by dissection technique (37.07) and aspiration (12.53).

Keywords
Oocyte grading; Percentage; Aspiration; Dissection; Slicing; Goat

Introduction
Reproduction is the basic field of live stock production. The genetic improvement of live stock can be obtained by maximum utilization of proven sires and dams by following the artificial insemination and Embryo Transfer Technology (ETT). Follicular oocytes could be matured in vitro and used for in vitro fertilization for producing large number of embryos (Agarawal, 1992; Suzuki et al., 1992). In vitro techniques are powerful tools for studying physiology of maturation, fertilization, development of pre-implantation embryos and increasing production as it gives access to micromanipulation of embryos. For such studies, a large number of in vitro produced embryos are needed, which in turn need larger number of oocytes only. The total number of oocytes obtained per ovary is varied with different techniques. Therefore, for efficient in vitro production of embryo from ovaries procured from abattoirs is necessary to develop technique that can maximize the oocyte recovery. In case of the goat, the first birth using the IVF procedure on ovulated oocytes was achieved by Hanada (1985) and in the last few years, many studies on in vitro maturation and IVF of goat oocytes were conducted.

1 Results and Discussion
1.1 Oocyte retrieval

The number of oocytes retrived was significantly higher in slicing method (5.52 ± 0.40), followed by dissection (2.52 ± 0.28) and aspiration (1.35 ± 0.18) (Figure 1). Similar techniques were also employed by Wani et al. (2000), Kumar et al. (2000), Gogoi et al. (2001) and Shirazi et al. (2005) with variable number or per cent of oocytes retrived in the ovaries of small ruminants.
  

 

Figure 1 Oocyte recovery


The different A, B, C and D grades of oocytes retrieved from left and right ovaries under aspiration, dissection and slicing techniques were not significantly differed between the numbers of oocytes except in B grade oocyte, significantly higher number was retrieved from right ovary under slicing technique (Figure 2). 

 

 

Figure 2 Grade of oocytes


The yield of grade A oocytes was not significantly differed between techniques except in slicing, The retrieval of grade B oocyte was significantly higher in dissection and slicing techniques rather than aspiration in the left ovary, while, significantly higher number was retrieved in slicing technique. The retrieval of oocytes of grades C and D significantly higher in slicing techniques only, from both left and right ovaries (Figure 3). 

 

 

Figure 3 Percent yield of different grade of oocyte


The present yield of oocyte was significantly higher in slicing (50.57) followed by dissection (37.52) and aspiration (20.70) techniques (Figure 3). However, there was no like wise, significantly higher per cent of A, B, C and D grade oocytes were retrieved in slicing techniques when compared to aspiration and dissection techniques.

Lorenzo et al. (1999) and recorded similar trend in per cent of oocyte retrieval Kumar et al. (2000), but vice versa was noticed by Bonde et al. (2000). 

1.2 Percent yield of oocytes
In the present study, B grade oocytes were retrieved to a greater (per cent) by aspiration (12.53), dissection (37.07) and slicing (50.57), while the recovery of grade D was found lowest by all the techniques. The aspiration technique also yielded grade A oocytes to a greater extent. The grade C oocytes were recovered to a greater extent by slicing technique. The overall yield was highest with slicing technique. The oocytes remain firmly attached to the small and medium sized follicles before cumulus expansion and cannot be aspired, but can be easily recovered from the small follicles when the slicing method is employed. Slicing of ovaries is a simple and efficient tool for recovering good quality oocytes, but the aspiration technique is laborious and time consuming. These reports also confirm the report of Pawshe et al. (1994). The grade A and B oocytes were retrieved greatly from aspiration technique and dissection technique when compared to grade C and D oocytes; the latter were recovered to a greater extent by slicing method. These findings were in confirmation with the report of Gogoi et al. (2001).

From the results of the present study, the maximum per cent yield of grade A and B oocytes was observed by aspiration technique and dissection technique, while the grade C and D oocytes were retrieved by slicing technique. Overall yield of oocytes was highest with slicing technique when compared to the other techniques.

2 Materials and Methods
The present study was undertaken during February to October months on local goat breeds that were maintained by the shepherd of Hyderabad, Andhra Pradesh. A total number of 120 non-gravid genitalia of local does were collected immediately after slaughter at municipal slaughter house and transported to the laboratory within 2 hr in a thermos at 34℃ in normal saline (0.9%) containing gentamicin (50 µg/mL). The genitalia were washed with tap water thrice and the ovaries were separated. The separated ovaries were sorted as left and right ovaries and washed separately with DPBS (pH 7.3) containing gentamicin (50 µg/mL).

2.1 Oocyte recovery
The oocytes were collected aseptically from the ovaries by three methods:

(1) Aspiration: The visible follicles present on the surface of the ovary were aspirated with 22 G needle fixed to 5 mL disposable syringe containing 1~2 mL of DPBS. The goat oocytes were aspirated from individual ovaries after carefully removing the extraneous tissue and were placed in petri dish containing 1 mL of DPBS for grading.

(2) Dissection Technique: The ovaries were placed in a sterile glass petri dish containing 2 mL of DPBS. All the visible follicles were carefully subjected to blunt dissection with the help of forceps and the remaining ovarian tissue was removed after a brief rinsing. The follicles were ruptured and the follicular fluid was allowed to flow into the DPBS.

(3) Slicing: The ovaries were held firmly with the help of forceps in a sterile glass petri dish containing 2 mL of DPBS. The ovaries were sliced into possible thin sections with a blade fixed to the artery forceps. The oocytes containing DPBS media were transferred to the petri dish and observed under Stereozoom microscope to grade the oocytes.

The petri dish was observed under Stereozoom microscope and the oocytes were transferred to a searching dish containing DPBS for grading.

2.2 Grading of oocytes
Oocytes were classified on the basis of cumulus layer as follows (Table 1; Figure 4) (Singh and Sharma, 1991).
 

 

Table 1 Classification grading of oocytes

 

 

Figure 4 A, B, C and D grades of oocytes in non descriptive dose


References

Agarawal K.P., 1992, In vitro maturation of caprineoocytes, Indian journal of Animal reproduction, 13(2): 195-197

Bonde S.W., Naqvi S.M.K., and Mittal J.P., 2000, Comparison of oocyte recovery from surface follicles of sheep and goat ovaries, International Journal of Animal Sciences 15(1): 1-4

Das G.K., Manjumdar A.C., and Gupta S.K., 1996, Study of collection techniques on oocyte recovery and quality in goat, International Journal Animal Science 11:143-144

Gogoi A.K., Borgohain B.N., Deka B.C., and Chakravarthy P., 2001, Comparative efficacy of aspiration and dissection techniques on the recovery of oocytes from goat ovaries, Indian Journal of Animal Reproduction 22: 19-22

Hanada A., 1985, In vitro fertilization in goats, Japan Journal of Animal Reproduction, 31: 21-26

Lorenzo P.L., Calduch T., Illera M.J., Illera J.C., Picazo R.A., and Illera M., 1999, Study of the ultra-structural morphology of sheep oocytes collected for in vitro maturation and fertilization, Medicina veterinaria 16(4): 196-201

Pawshe C.H., Totely S.M., and Jain S.K., 1994, A comparison of three methods of recovery of goat oocytes for in vitro maturation and fertilization, Theriogenology, 42(1): 117-125
http://dx.doi.org/10.1016/0093-691X(94)90668-9

Shirazi A., Shams-Esfandabadi N., and Hosseini S.M., 2005, A comparison of two recovery methods of ovine oocytes for in vitro maturation, Small Ruminant Research, 58(3): 283-286
http://dx.doi.org/10.1016/j.smallrumres.2004.11.002  

Singh R.B., and Sharma A., 1991, Importance of cumulus mass in the maturation of goat oocytes under in vitro conditions, National Symposium and 7th Annual Conference of Society of Animal Physiologists of India, Madras, pp.30

Kumar S., and Maurya S.N., 2000, Evaluation of oocyte retrieval methods from bubaline ovaries, Indian Journal of Animal Research, 34(2): 130-132

Suzuki T., Singh S.K., Sujata J., and Madam M.L., 1992, In vitro fertilization of water buffalo follicular oocytes and their ability to cleave in vitro, Theriogenology, 38(6): 1187-1194
http://dx.doi.org/10.1016/0093-691X(92)90130-J  

Wani N.A., Wani G.M., Khan M.Z., and Salahudin S., 2000, Effect of oocyte harvesting techniques on in vitro maturation and in vitro fertilization in sheep, Small Ruminant Research, 36: 63-67
http://dx.doi.org/10.1016/S0921-4488(99)00097-8

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