Small Ruminant Research 36 (2000) 63±67 Effect of oocyte harvesting techniques on in vitro maturation and in vitro fertilization in sheep N.A. Wani a,*, G.M. Wani a, M.Z. Khan a, S. Salahudin b a Division of Animal Reproduction, Gynecology and Obstetrics, Faculty of Veterinary Sciences and Animal Husbandry, S.K. University of Agricultural Sciences and Technology, PO.Box 953, G.P.O. 190001, Srinagar, India b Department of Immunology and Biochemistry, S.K. Institute of Medical Sciences, Soura, Srinagar, India Accepted 30 June 1999 Abstract Three different methods, viz., puncture, aspiration and slicing were used for harvesting the oocytes of ovine ovaries. The aim was to determine an ef cient method for oocyte harvesting and its effect on subsequent in vitro maturation and in vitro fertilization. The average total number of oocytes recovered per ovary was signi cantly higher by puncture (9.5 0.45) and slicing (9.5 0.40) than by the aspiration method (6.8 0.30) (P < 0.05). However, the percentage of good quality oocytes was higher in the aspiration method (64.4%), compared to the puncture (54.7%) or slicing (54.3%) methods. The oocytes obtained were matured and fertilized in vitro using the spermatozoa collected from the epididymus of slaughtered rams. There was no signi cant difference in the subsequent percentage of oocytes maturing and being fertilized in vitro between the three methods of oocyte harvesting. It can be concluded that all the three methods can be used for the collection of oocytes in sheep, without any detrimental effect on the subsequent maturation and fertilization rate. Also the spermatozoa collected from the epididymus of dead/slaughtered rams can be used successfully for the in vitro fertilization of oocytes in sheep. # 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Oocyte harvesting; In vitro maturation; In vitro fertilization 1. Introduction The number of high quality oocytes harvested from an ovary is an important consideration for the in vitro production of embryos. In bovine, the capacity to produce embryos by IVM/IVF has progressed very rapidly during the past few years (Leibo and Loskutoff, 1993). However, in sheep only a limited number of offspring have been produced using this technique. * Corresponding author. Tel.: 91-194-456150; fax: 91-194- 430590. Also in small ruminants a demand exists for basic research on zygote development and on the production of transgenic offspring. In vivo matured oocytes are obtained either by surgical or laparoscopic methods (Baldassare et al., 1994). These methods are expensive and the number of oocytes recovered per ovary is very small (Pawshe et al., 1994). Ovaries of slaughtered animals are the cheapest and most abundant source of primary oocytes for the large scale production of embryos through IVM/IVF procedures (Agrawal et al., 1995). Follicular dissection was rst used to recover ovine follicular 0921-4488/00/$ ± see front matter # 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0921-4488(99)00097-8
64 N.A. Wani et al. / Small Ruminant Research 36 (2000) 63±67 oocytes (Crosby et al., 1981; Fukui et al., 1988). Currently slicing (Wahid et al., 1992a,b) and aspiration of follicles (Slavik et al., 1992; Watson et al., 1994) are routinely used for oocyte recovery in ovine. The objective of the study was to evaluate three techniques of oocyte recovery from slaughtered sheep ovaries and the effect on in vitro maturation and in vitro fertilization. 2. Materials and methods Ovaries were collected from a local slaughter house in Srinagar city and transported to the laboratory within 3±4 h of slaughter in dulbeccos phosphate buffer saline (DPBS) in a thermos ask. In the laboratory each ovary was freed from the surrounding tissues and overlying bursa. Each ovary was treated to three washings in DPBS and two washings in oocyte harvesting medium (DPBS 4 mg/ml BSA 1 50 IU/ml Penicillin 2 ). Each ovary was processed individually and the oocytes harvested by one of the following three techniques: Puncture: Ovaries were placed in a pertridish containing 5 ml of oocyte harvesting medium, held with the help of forceps and the whole ovarian surface was punctured with an 18 gauge hypodermic needle. Slicing: Ovaries were placed in a petridish containing 5 ml of the oocyte harvesting medium, held with the help of forceps. Incisions were given along the whole ovarian surface using a scalpel blade. Aspiration: Visible follicles were aspirated using a 20 gauge hypodermic needle attached with a sterile disposable syringe containing 2 ml harvesting medium. The media along with the collected oocytes was then transfered to a 35 mm petridish. While harvesting the oocytes by puncture and slicing methods, the ovary was kept completly dipped in the medium. In all the three techniques, the petridishes were kept undisturbed for 5 min, allowing the oocytes to settle down. Excess media was taken out by a syringe without disturbing the oocytes art the bottom of petridish. The petridishes were then examined under an inverted microscope, and the total number of oocytes harvested, counted. The oocytes were graded as good, fair and poor on the basis of cumulus cells and cytoplasm. Good: Oocytes with many complete layers of cumulus cells and uniform cytoplasm. Fair: Oocytes with thin or incomplete layers of cumulus cells and uniform cytoplasm. Poor: Oocytes with few or no cumulus cells. The number of good, fair and poor oocytes obtained were recorded for each ovary. In vitro maturation: The good and fair oocytes were pooled and subject to two to three washings in maturation medium, composed of TCM-199 3 supplemented with 10% fetal calf serum 4, 0.1 IU/ml Human menopausal gonadotrophin 5 and 50 IU/ml Penicillin. Ten oocytes were placed in 2 ml maturation medium and incubated at 38.58C under 5% CO 2 with a saturated humidity for 24±26 h. After the end of incubation a portion of oocytes was freed from the cumulus cells by continuously pippeting in and out of a capillary, so that completely denuded oocytes were obtained. The denuded oocytes were xed in an ethanol and acetic acid solution (3 : 1) for 24 h, stained with a 1% aceto orecine stain and examined under a high power microscope. The maturation state of oocytes was evaluated on the basis of nuclear maturation. The oocytes were classi ed as: germinal vesicle (GV); germinal vesicle breakdown (GVB); metaphase-i (M-I); metaphase-ii (M-II); degenerated (Dg.). In vitro fertilization: Sheep tests obtained from the abattoir, were washed with saline and the cauda epididymus isolated. The area below the deferent duct was located and presumed to contain mature sperm. This area was trimmed free of the covering tissues. An incision was made on the convoluted tubules with a sterile hypodermic needle. The gushing uid, rich in sperm, was collected with the aid of a micropipete and mixed with 4 ml washing medium. The sperm were given two washings by centrifugation at 1500 rpm for 10 min using TCM-199, supplemented with 4 mg/ml BSA and 50 IU/ml Penicillin. 100 ml of the loose semen pellet was covered by 2 ml of fertilization medium, composed of TCM-199 supplemented with 4 mg/ml BSA, 50 IU/ml Penicillin and 50 IU/ml 1 Sigma, USA. 2 Serva Feinbiochemica, Heidelberg. 3 Himedia, Mumbai, India. 4 Sigma, USA. 5 Pergonal, Serono Switzerland.
N.A. Wani et al. / Small Ruminant Research 36 (2000) 63±67 65 Table 1 Effect of three different harvesting techniques on oocyte recovery in sheep Method of collection Total No. of ovaries Oocytes / ovary (mean S.E.) a Total Good (%) Fair (%) Poor (%) Puncture 47 9.5 0.45a 5.2 0.25 2.0 0.17 2.2 1.09 (54.7) (21.5) (23.8) Slicing 61 9.5 0.4a 5.2 0.23 2.2 0.16 2.2 1.07 (54.3) (22.8) (22.9) Aspiration 51 6.8 0.31b 4.4 0.25 1.5 0.13 1.0 0.09 (64.4) (22.4) (13.2) a Mean values in the same column with different alphabets differ signi cantly at P < 0.05. heparin, and kept in a CO 2 incubator (38.5 o C ) at an angle of 458. The sperm was allowed to swim up for 2 h. The matured oocytes were washed once in fertilization medium and then transferred to 2 ml of fertilization medium. The highly motile spermatozoa from the upper layers were added to the oocytes at the concentration of 1±2 10 6 /ml approximately. The mixture of gametes was incubated for 18±22 h. The oocytes were xed in acid and alcohol for 24 h, stained with a 1% aceto±orcine stain and examined under high power microscope for the sperm penetration and fertilization. The sperm in perivitalline space or in vitellus, swollen sperm head in ooplasm or a male and female pronuclei were observed and recorded. The data obtained was analyzed as per the standard analytical procedures (Snedecor and Cochran, 1967). 3. Results 3.1. Effect of harvesting technique on oocyte recovery The total number of oocytes recovered per ovary by puncture (9.5 0.45) and slicing (9.5 0.4) were signi cantly higher than by aspiration (6.8 0.3) method. The puncture and slicing method yielded approximately similar numbers of oocytes. The percentage of good oocytes was however, higher for the aspiration method (64.4), compared to the (54.7) and slicing (54.3) methods (Table 1). In vitro maturation: Oocytes recovered by puncture, slicing and aspiration were matured in vitro and their nuclear maturation evaluated. The maturation rate of oocytes collected by puncture, slicing and aspiration was 62.5%, 61.7% and 66.3%, respectively (Table 2). In vitro fertilization: The penetration rates were 52.0, 54.4 and 51.3, respectively, for oocytes obtained by the puncture, slicing and aspiration methods (Table 3). 4. Discussion The quantity and quality of oocytes recovered per ovary has been an important consideration in the production of IVM±IVF embryos. Several methods have been used for the collection of oocytes from the ovaries of cattle (Kataska, 1984), goats (Mogas et al., Table 2 Effect of harvesting technique on in vitro maturation of oocytes in sheep Method of collection Total No. of oocytes Maturation (%) Degenerated GV GVB M-I M-II Puncture 80.0 9.0 3.0 12.0 50.0 6.0 (11.2) (3.7) (15.0) (62.5) (7.5) Slicing 94.0 7.0 4.0 20.0 58.0 5.0 (7.4) (4.3) (21.3) (61.7) (5.3) Aspiration 86.0 5.0 3.0 14.0 57.0 7.0 (5.8) (3.5) (17.4) (66.3) (8.1)
66 N.A. Wani et al. / Small Ruminant Research 36 (2000) 63±67 Table 3 Effect of harvesting technique on in vitro fertilization of oocytes in sheep Method of collection Total No. of oocytes No. of oocytes penetrated (%) No. of oocytes with polyspermy (%) Puncture 73.0 38.0 (52.0) 1.0 (1.4) Slicing 101.0 55.0 (54.4) 2.0 (1.98) Aspiration 76.0 39.0 (51.3) 2.0 (2.6) obtained in goats (Pawshe et al., 1994). No signi cant difference was found in the maturation and fertilization rates of oocytes obtained by the three different techniques. However, puncture of ovarian surface by an 18 gauge hypodermic needle was found to be simple practical and more ef cient method for collecting oocytes in sheep, compared to the slicing method. References 1992; Pawshe et al., 1994) and sheep (Wahid et al., 1992a,b). The most commonly used recover methods in sheep are slicing (Wahid et al., 1992a,b), aspiration of visible follicles (Watson et al., 1994) or follicular dissection (Fukui et al., 1988). In the present study the oocytes were collected from the ovaries by three different methods and the effect on in vitro maturation and in vitro fertilization evaluated. Puncture and slicing techniques yielded a higher number of oocytes per ovary than the aspiration methods (P < 0.05). Slicing and puncture yielded similar numbers of oocytes, however, the slicing method resulted in production of more debris which interfered in the search of oocytes under microscope. Observations in the present study are comparable to those reported for cattle (Iwasaki et al., 1987; Kataska, 1984; Lonergan et al., 1991), goats (Mogas et al., 1992; Pawshe et al., 1994) and sheep (Wahid et al., 1992a,b) and demonstrated that the puncture of whole ovarian surface by an 18 gauge hypodermic needle is a simple and ef cient method of recovering a high number of morphologically normal oocytes. It is also comparable to the slicing technique. The lower number of oocytes recovered by the aspiration method may be attributed to the presence of some follicles embedded deeply within the cortex, which are released by puncture or slicing of the ovary. Some of the oocytes may even be lost during aspiration of follicles, which is not possible when using the slicing or puncture methods. The good and fair quality oocytes obtained were matured in vitro. There was no signi cant difference in the number of oocytes reaching metaphase-ii. Similar results were also observed in cattle (Carolon et al., 1992), goats (Pawshe et al., 1994) and sheep (Wahid et al., 1992a,b). Fertilization rates were not signi cant different and can be compared with the results Agrawal, K.P., Sharma, T., Sexena, C., Sharma, N., 1995. Chronology of rst meiotic events of caprine oocytes matured in vitro. Ind. J. Anim. Sci. 65, 285±288. Baldassare, H., Matos, D.G.D.E., Furnus, C.C., Castro, T.E., Fischer, E.I.C., 1994. Technique for ef cient recovery of sheep oocytes by laproscopic folliculocentesis. Anim. Reprod. Sci. 35, 145±150. Carolon, C., Monaghan, P., Mehmood, A., Lonergan, P., Gallagher, M., Gordon, I., 1992. Slicing of bovine ovaries as a means of oocyte recovery. J. Reprod. Fertil. 9, 51. Crosby, I.M., Osborn, J.C., Moor, R.M., 1981. Follicle cell regulation of protein synthesis and developmental competence in sheep oocytes. J. Reprod. Fertil. 62, 575±582. Fukui, Y., Glew, A.M., Gandol, F., Moor, R.M., 1988. In vitro culture of sheep oocytes matured and fertilized in vitro. Theriogenology 29, 883±891. Iwasaki, S., Kono, T., Nakahai, T., Shioyo, Y., Fukushima, M., Hanada, K., 1987. New methods for the recovery of oocytes from the bovine ovarian tissue in relation to in vitro maturation and fertilization. Jpn. J. Anim. Reprod. 33, 188±191. Katska, L., 1984. Comparison of two methods for the recovery of ovarian oocytes from slaughtered cattle. Anim. Reprod. Sci. 7, 461±463. Leibo, S.P., Loskutoff, N.M., 1993. Cryobiology of in vitro derived bovine embryos. Theriogenology 39, 81±94. Lonergan, P., Vergos, E., Kinis, A., Shrif, H., Gallagher, M., Gordon, I., 1991. The effect of recovery method on type of bovine oocytes obtained for in vitro maturation. Theriogenology 35, 231. Mogas, T., Martino, A., Palamo, M.J., Paramio, M.T., 1992. Effect of method of recovery on the number and type of oocytes obtained for IVM. J. Rerod. Fertil. 9, 52. Pawshe, C.H., Totey, S.M., Jain, S.K., 1994. In uence of the methods of recovery of goat oocytes for in vitro maturation and fertilization. Theriogenology 42, 117±125. Slavik, T., Fulka, J., Gall, I., 1992. Pregnancy rate after the transfer of sheep embryos originated from randomly chosen oocytes matured and fertilized in vitro. Theriogenology 38, 749±756. Snedecor, G.W., Cochran, W.G., Statistical methods, Oxford and IBH publishing company, New Delhi, 1967. Wahid, H., Gordon, I., Sharif, H., Lonergan, P., Gallagher, M., 1992a. Effect and ef ciency of recovery method for obtaining ovine follicular oocytes for in vitro procedures. Theriogenology 37, 318.
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