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In vitro maturation of sheep oocytes in different media during breeding and non-breeding seasons
Small Ruminant Research 43 (2002) 31±36
In vitro maturation of sheep oocytes in different media during breeding and non-breeding seasons B.S. Rao, K.S. Naidu, D. Amarnath, R. Vagdevi, A.S. Rao, K.V. Brahmaiah, V.H. Rao* Embryo Biotechnology Laboratory, College of Veterinary Science, Tirupati 517 502, India Accepted 29 June 2001
Abstract Sheep oocytes were aspirated from ovaries collected from a commercial slaughter house during the breeding and nonbreeding seasons. The mean number of oocytes collected per ovary and the proportions of acceptable or poor oocytes were similar between the seasons. In vitro maturation (IVM) of sheep oocytes was evaluated during breeding and non-breeding seasons in medium 199 supplemented with follicle stimulating hormone (FSH), lutinizing hormone (LH) and estradiol, bovine embryonic ¯uid, estrus sheep serum or ovine follicular ¯uid. IVM was assessed by extrusion of the ®rst polar body and nuclear maturation to metaphase II by staining with Hoechst 33342. There was no difference in ef®ciency of IVM of oocytes collected during the breeding and non-breeding seasons. Medium 199 supplemented with estrus sheep serum supported better rates of IVM. In a separate series of experiments four differently supplemented media and monolayers of sheep granulosal cell culture were investigated for their ability to support IVM of sheep oocytes collected during the breeding season. The results indicated that estrus sheep serum supplementation and granulosal cell monolayers were the best. Bovine embryonic ¯uid and ovine follicular ¯uid supported similar rates of IVM, which were lower than those supported by estrus sheep serum and granulosal cell monolayers but higher than the control medium. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Oocytes; Sheep; In vitro maturation; Breeding season
1. Introduction Prominent breeds of sheep in southern parts of India are the Nellore, Mandya, and Deccani. These animals do not exhibit estrus during the months of March± April and September±October, considered the nonbreeding season (Kaushish, 1994). However, in our laboratory, the presence of a good number of antral follicles on the ovaries of sheep is frequently observed *
Corresponding author. Tel.: 91-8574-49376; fax: 91-8574-49563. E-mail address: [email protected] (V.H. Rao).
(unpublished) even during the non-breeding season. Information on the ability of oocytes collected from antral follicles during the non-breeding season to undergo in vitro maturation (IVM) is lacking. Therefore, the ability of sheep follicular oocytes collected during the breeding and non-breeding seasons to undergo IVM in four different culture media was investigated. Further the supplementation of IVM medium with bovine embryonic ¯uid (a commercially available serum substitute), estrus sheep serum, ovine follicular ¯uid and monolayers of granulosal cell cultures (GCC) to support IVM of sheep oocytes collected during the breeding season was assessed.
0921-4488/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 0 1 ) 0 0 2 5 4 - 1
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B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36
2. Materials and methods 2.1. General Two separate experiments on IVM of sheep follicular oocytes were undertaken. The ®rst experiment compared the ability of sheep follicular oocytes collected during the breeding and non-breeding seasons to undergo successful IVM in differently supplemented media. The second experiment compared the in¯uence of bovine embryonic ¯uid (BEF), estrus sheep serum (ESS), ovine follicular ¯uid (OFF), and monolayers of GCC on IVM of sheep oocytes collected during breeding season. Medium 199 (TCM 199) supplemented with hormones served as the control. 2.2. Preparation of media All the media, hormones, fetal calf serum (FCS) and other chemicals used were purchased from Sigma Chemical Co., USA and plastics from Nunclon, Denmark. HEPES buffered tissue culture medium 199 (TCM 199H) supplemented with 10% FCS, 25 IU heparin and 50 mg/ml of gentamycin sulfate was used for handling oocytes prior to and after culture. Control medium was prepared by supplementing bicarbonate buffered TCM 199 (TCM 199B) with 5 mg/ml each of follicle stimulating hormone (FSH), lutenizing hormone (LH), 1 mg/ml of estradiol and 50 mg/ml of gentamycin sulfate. Control medium was additionally supplemented with 20% (v/v) BEF, OFF or ESS for IVM of oocytes. TCM 199B supplemented with FCS 10% (v/v), gentamycin sulfate 50 mg/ml, streptomycin 0.1 mg/ml and penicillin 100 IU/ml for culture of granulose cells. All media were sterilized by ®ltration through 0.22 mm ®lters (Sartorius, Germany) and equilibrated with 5% carbon dioxide in air in a humidi®ed atmosphere at 38.5 8C for at least 2 h. 2.3. Collection and processing of biological materials Sheep ovaries were collected from ewes of mixed breeds from a slaughter house and transported to the laboratory, a distance of about 200 km, within 5±6 h in warm (35 8C) phosphate buffered saline (PBS) supplemented with penicillin 1000 IU/ml and streptomy-
cin 1000 mg/ml. In the laboratory, the antral follicles (2±5 mm in diameter) on the ovaries were aspirated using a 22-gauge needle attached to a plastic 5 ml syringe containing 1 ml of oocyte handling medium. The cumulus oocyte complexes (COC) were isolated under a stereo zoom microscope (Nikon Corporation, Japan) and graded as good, fair or poor (Fig. 1A±C). Only good and fair oocytes were considered acceptable and used in IVM experiments. Follicular ¯uid was aspirated from transparent and healthy looking ovarian follicles greater than 4 mm in diameter. Pooled follicular ¯uid samples were centrifuged at 2000 rpm for 10 min. Supernatant was carefully removed and re-centrifuged as above. Finally the supernatant was ®ltered and stored at 20 8C in 1 ml aliquots until used. On the day of use, the follicular ¯uid was heated for 30 min at 56 8C in a water bath to inactivate the complement. Blood from jugular veins of ewes on the second day of estrus was collected in sterile glass tubes and was allowed to clot for 1±2 h at room temperature (25 8C). After the clot formed, the tubes were transferred to a refrigerator (4±5 8C) and the serum was allowed to separate. The serum was carefully collected and centrifuged at 1000 rpm for 10 min. The serum was sterilized by ®ltration and the complement was ®xed and stored until used. Follicles more than 4 mm in diameter were ¯ushed with 1 ml of Hanks balanced salt solution (HBSS) without calcium and magnesium using a 23-gauge needle attached to 2 ml plastic syringe. The aspirate was transferred to a test tube and 1 ml of hyaluronidase (150 IU/ml) was added to avoid coagulation. The contents were then centrifuged for 10 min at 2000 rpm. The supernatant was removed, the clot was re-suspended in TCM 199H and re-centrifuged. Finally the clot was diluted in 1 ml of granulosal cell culture medium. A 5 ml sample of this cell suspension was used to estimate the concentration and proportion of live and dead cells using Trypan blue stain following the procedure provided by the manufacturer. The cell concentration was adjusted to 2 106 cells/ml by adding required quantity of culture medium. To establish monolayers 500 ml of this suspension was placed in each of the four wells of a multidish (Nunclon, Denmark) and cultured in 5% carbon dioxide in air at 38.5 8C for 5±6 days. 0.25 ml of the culture medium was replaced with fresh medium every 48 h.
B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36
33
Fig. 1. In vitro maturation of sheep oocytes. (A) Freshly collected good oocyte; (B) freshly collected fair oocyte; (C) freshly collected poor oocyte; (D) in vitro matured oocyte exhibiting ®rst polar body; (E) germinal vesicle stage; (F) germinal vesicle break down; (G) metaphase I; (H) anaphase; (I) telophase; (J) metaphase II.
Light weight mineral oil equilibrated with the appropriate culture medium overnight in a humidi®ed atmosphere of 5% carbon dioxide in air at 38.5 8C was used to cover the micro-drops of oocyte cultures. Good and fair COC were washed twice in oocyte handling medium and an additional two times in the appropriate maturation medium. Each day, 5±10 COC collected were randomly allotted to each of the experimental groups. However, on certain days some of the experimental groups were not
allotted any oocytes (Table 1). For studying the in¯uence of various treatments, 5±10 COC were placed in 50 ml micro-drops of differently supplemented culture media including control or on monolayers of granulosal cells. Oocytes in micro-drops were covered with mineral oil and cultured for 24 h at 38.5 8C under humidi®ed atmosphere of 5% carbon dioxide in air. Oocytes on granulosal cell layers were similarly incubated but without mineral oil covering.
34
B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36
Table 1 In vitro maturation of sheep oocytes in different media during breeding seasona Culture medium
BEF ESS OFF GCC Control Fresh a
No. of oocytes stained (replicates)
No. of oocytes in different nuclear maturation stages (% of stained)
130 137 131 134 106 117
100 (77) 118 (86) 100 (76) 110 (82) 61 (58) NIL
(18) (19) (18) (17) (17) (15)
M II b c b b a
MI
A/T
GVBD
GV
16 (12) b 9 (7) a 12 (9) a 11 (8) a 14 (13) a NIL
7 7 13 10 12 2
1 1 1 1 3 22
6 2 5 2 16 93
(6) ab (6) ab (10) bc (8) ab (11) c (2) a
(0.8) a (0.7) a (0.8) a (0.8) a (3) b (19) c
(5) a (2) a (4) a (2) a (15) b (80) c
Values within a column with different letters differ at P < 0:01.
2.4. Evaluation of oocyte maturation At the end of IVM period oocytes from different groups were separately denuded of cumulus cells by repeatedly passing through a narrow bore glass pipette. Samples were washed in Hoechst 33342 ¯uorescent dye (5 mg/ml) and incubated in the dye for 15 min at 38.5 8C. Subsequently oocytes were examined on an inverted microscope (Leica, Germany) for extrusion of ®rst polar body (Fig. 1D) and under ¯uorescent light for nuclear maturation. Nuclear maturation stages were germinal vesicle (GV), germinal vesicle break down (GVBD), metaphase I, anaphase I, telophase I and metaphase II (Fig. 1E±J). 2.5. Statistical analysis The data on mean oocytes collected per ovary and the proportions of good, fair and poor oocytes (Table 2) were analyzed by normal deviate test. Rates of IVM were analyzed using a mathematical model that included the ®xed effect due to culture medium (BEF, ESS, OFF and control) and breeding season (breeding and non-breeding) and residual error.
Data on IVM of oocytes in different media during breeding season (experiment 2) was analyzed using a mathematical model that included ®xed effect due to culture medium (BEF, ESS, OFF, GCC, control and fresh) and residual error. Signi®cance of difference was determined by Duncan's multiple range test (Snedecor and Cochran, 1989). 3. Results A total of 1281 sheep ovaries were collected at the slaughter house which resulted in 1691 oocytes (Table 2) during the breeding and non-breeding seasons. There was no difference between seasons in the mean number of oocytes recovered per ovary. The proportions of oocytes in different quality groups were similar between the breeding and non-breeding seasons. The proportions of oocytes exhibiting ®rst polar body after IVM in three different media during the breeding and non-breeding seasons (BEF: 30:7 3:39 versus 30:5 1:89; ESS: 30:6 6:65 versus 37:7 5:95; OFF: 33:7 7:74 versus 28:4 4:25) were
Table 2 Collection of ovarian follicular oocytes in sheepa Source No. No. No. No. No. No.
of of of of of of a
ovaries oocytes oocytes/ovary good oocytes (%) fair oocytes (%) poor oocytes (%)
Breeding season
Non-breeding season
Total
824 1180 1.43 0.55 a 491 (42) b 404 (34) c 285 (24) d
457 511 1.12 0.45 a 200 (39) b 165 (32) c 146 (29) d
1281 1691 1.32 0.50 691 (41) 569 (34) 431 (26)
Values with same letters within a row do not differ at P < 0:05.
B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36 Table 3 In vitro maturation of sheep oocytes in different media during two different seasonsa Culture medium
BEF (20% (v/v)) ESS (20% (v/v)) OFF (20% (v/v)) Control
Percentage oocytes in metaphase II S.E. (replicates) Non-breeding season
Breeding season
59.3 69.3 56.7 45.4
63.4 3.27 a (8) 71.4 2.69 b (8) 59.2 2.88 a (8) 47.6 3.18 c (8)
3.03 2.94 2.18 3.01
a (8) b (8) a (8) c (8)
a Values with same letters within a row do not differ at P < 0:05, but values within a column with different letters differ at P < 0:01.
similar (P < 0:05). The interaction between season and the medium was also not signi®cant. The in¯uence of different media on proportion of oocytes maturing to M II stage was not signi®cantly different between the two seasons (Table 3) nor was the interaction between season and treatment. However, different media used had supported in vitro maturation at signi®cantly different rates (Table 3). Thus, ESS had supported the highest rate of IVM during both the seasons. BEF and OFF supported similar rates of IVM which were lower than that supported by ESS but higher than the control. It is interesting to note that among freshly collected COC, only 80% during the breeding season (Table 1) and 86% (data not shown) during non-breeding season were at the GV stage (P < 0:05). During the breeding season the proportions of oocytes exhibiting ®rst polar body after IVM in ESS and GCC were equal (38:8 2:98 versus 39:7 3:61) and signi®cantly higher than in all other groups (BEF: 28:6 2:12; OFF: 28:3 2:43; control: 25:6 2:85). Similarly ESS and GCC supported the highest rate of IVM to M II stage followed by BEF and OFF, and the control with the lowest rate (Table 1). Signi®cantly higher proportions of oocytes remained in GV and GVBD stages during maturation in control medium. 4. Discussion The mean rate of recovery of oocytes at 1.32 per ovary obtained in this study is lower than 4 oocytes per ovary reported by Wahid et al. (1992), 2.17 by Datta et al. (1993) and 2.75 by Das et al. (1996). However, in
35
these reports, the number of ovaries used were relatively small (44±140 versus 1281 in the present study). Further Wahid et al. (1992) also reported that only 1.5 oocytes per ovary were obtained when the aspiration time per ovary was reduced to 1.03 min. After transporting the ovaries from a slaughterhouse located 200 km away it was necessary to aspirate the oocytes in the shortest possible time before culture. This may have reduced the oocyte recovery rate. The number and quality of oocytes obtained (Table 2) does not support differential folliculogenic activity of the ovaries during the breeding and nonbreeding seasons. It is, however, possible that while ovarian follicular growth does not differ between seasons, the amount of estrogens produced by the ovarian follicles may differ between the seasons resulting in the observed differences (Kaushish, 1994) in occurrence of estrus. Estimation of estrogen content of sheep ovarian follicles during the breeding and non-breeding seasons could clarify this. Alternatively, antral follicles that appear on ovaries during the non-breeding season may not grow to ovulatory size to produce suf®cient estrogens. The ability of oocytes to undergo IVM as assessed by extrusion of ®rst polar body and development to M II (Table 3) did not differ signi®cantly between the seasons but was dependent on the medium. In a separate series of experiments during the breeding season, ESS and GCC supported the highest rate of IVM (Table 1). It has been reported that the addition of estrus cow serum to culture medium supported high rates of IVM of sheep oocytes (Yadav et al., 1997). Estrus goat serum (Mogas et al., 1995, 1997) and estrus buffalo serum (Madan et al., 1994) were reported earlier to support high rates of IVM of oocytes from respective species. BEF is tested as a commercial serum substitute to support IVM of sheep oocytes. In the present study, BEF supported IVM of oocytes to a lesser extent than ESS and GCC. Nevertheless, it was better than the control medium. This is in contrast to the observation on goat oocytes (Devaraju et al., 1997), where BEF and GCC supported similar rates of IVM. The ability of BEF to support IVM of sheep oocytes may vary with species. In the present study, sheep follicular ¯uid supported lower IVM rate than ESS and GCC. In the only earlier report on the use of sheep follicular ¯uid, Sun et al.
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B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36
(1994) observed a higher rate of IVM with OFM from follicles 6 mm or more in diameter. Follicular ¯uid collected from small follicles was reported to support lower rates of IVM (Sun et al., 1994). The relatively smaller size of the follicles from which the OFF was collected in the present study may have contributed to lower frequency of IVM. The IVM rates of 58±86% in different media were lower compared to published reports (Brun et al., 1986; Salykbaev et al., 1986; Agrawal and Polge, 1988; Szollosi et al., 1988; Holm et al., 1991; Sun et al., 1994; Baldassarre et al., 1994; Byrd et al., 1997). However, in the present study, 80% of freshly collected oocytes were in GV stage (Table 1) suggesting approximately 20% of oocytes collected were abnormal and had no ability to undergo IVM. Thus, the IVM rate may be adjusted for a 20% reduction in oocytes used. On this basis the IVM rate in this experiment would be between 72 (control) and 100% (ESS) which is comparable to the reports from other laboratories. 5. Conclusions The follicular oocytes collected from south Indian sheep both during the breeding and non-breeding seasons were similar in their ability to undergo IVM. Furthermore, higher rates of in vitro maturation of sheep oocytes may be possible by supplementation of TCM 199B with hormones and estrus sheep serum or on monolayers of granulosa cell culture. Acknowledgements This work was supported by a research grant from the ANGR Agricultural University to V.H. Rao, Graduate studies scholarship of the Government of Andhra Pradesh, India to B.S. Rao and junior research fellowship the Department of Biotechnology, Government of India to R. Vagdevi. We are grateful to Drs. K. Rekkas and S. Belibasaki for critical review of the manuscript and constructive suggestions. References Agrawal, K.P., Polge, C., 1988. In vitro maturation of sheep oocytes. Indian J. Anim. Reprod. 9, 8±9.
Baldassarre, H., Castro, T.E., Furnus, C.C., de Matos, D.G., 1994. In vitro maturation and fertilisation of sheep oocytes collected by laparoscopic folliculocentesis. Theriogenology 41, 159. Brun, J., Iritani, A., Leidl, W., 1986. Fertilisation in vitro of ovine follicular oocytes with ejaculated spermatozoa capacitated in a chemically de®ned medium. Zuchtygiene 21, 59±63. Byrd, S.R., Flores-Foxworth, G., Applewhite, A.A., Westhusin, M.E., 1997. In vitro maturation of ovine oocytes in a portable incubator. Theriogenology 47, 857±864. Das, S.K., Chauhan, M.S., Palta, P., Katiyar, P.K., Madan, M.L., 1996. Replacement of fetal bovine serum and FSH with buffalo follicular ¯uid in in vitro maturation of buffalo oocytes. Theriogenology 45, 245. Datta, T.K., Goswami, S.L., Das, S.K., 1993. Comparative ef®ciency of three oocyte recovery methods from sheep ovaries. Indian J. Anim. Sci. 63, 1178±1179. Devaraju, B., Naidu, K.S., Amarnath, D., Rao, K.V., Rao, V.H., 1997. Comparison of the effect of a commercial serum substitute and goat follicular ¯uid on nuclear maturation of goat oocytes. Theriogenology 47, 189. Holm, P., Irvine, B., Armstrong, D.T., Seamark, R.F., 1991. In vitro production of sheep blastocysts from IVM oocytes using frozen semen and oviduct epithelial cell co-culture for IVF. Theriogenology 35, 214. Kaushish, S.K., 1994. Sheep Production in the Tropics and Subtropics. Scienti®c Publishers, Jodphur, India, pp. 26±28. Madan, M.L., Singla, S.K., Chauhan, M.B., Manik, R.S., 1994. In vitro production and transfer of embryos in buffaloes. Theriogenology 41, 139±143. Mogas, T., Izquierdo, M.D., Palomo, M.J., Aramio, M.T., 1995. Effect of hormones serum source and culture system on the IVM and IVF of prepubertal goat oocytes and subsequent embryo development. Theriogenology 43, 284. Mogas, T., Palomo, M.J., Iqzuieriz, M.D., Paramio, M.T., 1997. Developmental capacity of in vitro matured and fertilised oocytes from prepubertal and adult goats. Theriogenology 47, 1189±1203. Salykbaev, T., Dombroskii, N.N., Dzhienbaeva, R.S., Murazamadiev, A.M., 1986. Ability of oocytes from sheep of different ages to mature when incubated within the follicles. Eksperimential Noi Biologii Akademiya Nauk, Kazakhskoi SSR 19, 167±169. Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods. Iowa State University Press, Ames, Iowa, USA. Sun, F.J., Holm, P., Irvine, B., Seamark, R.F., 1994. Effect of sheep and human follicular ¯uid on the maturation of sheep oocytes in vitro. Theriogenology 41, 981±988. Szollosi, D., Deslmedi, V., Crozet, N., Drender, C., 1988. In vitro maturation of sheep ovarian oocytes. Reprod. Nutr. Develop. 13, 1047±1080. Wahid, H., Gordan, I., Sharif, H., Lonergan, P., Monaghan, P., Gallagher, M., 1992. Effect and ef®cacy of recovery methods for obtaining ovine follicular oocytes for in vitro procedures. Theriogenology 37, 318. Yadav, B.R., Katiyar, R.K., Chauhan, M.S., Madan, M.L., 1997. Chromosome con®guration during in vitro maturation of goat, sheep and buffalo oocytes. Theriogenology 47, 943±951.
In vitro maturation of sheep oocytes in different media during breeding and non-breeding seasons B.S. Rao, K.S. Naidu, D. Amarnath, R. Vagdevi, A.S. Rao, K.V. Brahmaiah, V.H. Rao* Embryo Biotechnology Laboratory, College of Veterinary Science, Tirupati 517 502, India Accepted 29 June 2001
Abstract Sheep oocytes were aspirated from ovaries collected from a commercial slaughter house during the breeding and nonbreeding seasons. The mean number of oocytes collected per ovary and the proportions of acceptable or poor oocytes were similar between the seasons. In vitro maturation (IVM) of sheep oocytes was evaluated during breeding and non-breeding seasons in medium 199 supplemented with follicle stimulating hormone (FSH), lutinizing hormone (LH) and estradiol, bovine embryonic ¯uid, estrus sheep serum or ovine follicular ¯uid. IVM was assessed by extrusion of the ®rst polar body and nuclear maturation to metaphase II by staining with Hoechst 33342. There was no difference in ef®ciency of IVM of oocytes collected during the breeding and non-breeding seasons. Medium 199 supplemented with estrus sheep serum supported better rates of IVM. In a separate series of experiments four differently supplemented media and monolayers of sheep granulosal cell culture were investigated for their ability to support IVM of sheep oocytes collected during the breeding season. The results indicated that estrus sheep serum supplementation and granulosal cell monolayers were the best. Bovine embryonic ¯uid and ovine follicular ¯uid supported similar rates of IVM, which were lower than those supported by estrus sheep serum and granulosal cell monolayers but higher than the control medium. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Oocytes; Sheep; In vitro maturation; Breeding season
1. Introduction Prominent breeds of sheep in southern parts of India are the Nellore, Mandya, and Deccani. These animals do not exhibit estrus during the months of March± April and September±October, considered the nonbreeding season (Kaushish, 1994). However, in our laboratory, the presence of a good number of antral follicles on the ovaries of sheep is frequently observed *
Corresponding author. Tel.: 91-8574-49376; fax: 91-8574-49563. E-mail address: [email protected] (V.H. Rao).
(unpublished) even during the non-breeding season. Information on the ability of oocytes collected from antral follicles during the non-breeding season to undergo in vitro maturation (IVM) is lacking. Therefore, the ability of sheep follicular oocytes collected during the breeding and non-breeding seasons to undergo IVM in four different culture media was investigated. Further the supplementation of IVM medium with bovine embryonic ¯uid (a commercially available serum substitute), estrus sheep serum, ovine follicular ¯uid and monolayers of granulosal cell cultures (GCC) to support IVM of sheep oocytes collected during the breeding season was assessed.
0921-4488/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 0 1 ) 0 0 2 5 4 - 1
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B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36
2. Materials and methods 2.1. General Two separate experiments on IVM of sheep follicular oocytes were undertaken. The ®rst experiment compared the ability of sheep follicular oocytes collected during the breeding and non-breeding seasons to undergo successful IVM in differently supplemented media. The second experiment compared the in¯uence of bovine embryonic ¯uid (BEF), estrus sheep serum (ESS), ovine follicular ¯uid (OFF), and monolayers of GCC on IVM of sheep oocytes collected during breeding season. Medium 199 (TCM 199) supplemented with hormones served as the control. 2.2. Preparation of media All the media, hormones, fetal calf serum (FCS) and other chemicals used were purchased from Sigma Chemical Co., USA and plastics from Nunclon, Denmark. HEPES buffered tissue culture medium 199 (TCM 199H) supplemented with 10% FCS, 25 IU heparin and 50 mg/ml of gentamycin sulfate was used for handling oocytes prior to and after culture. Control medium was prepared by supplementing bicarbonate buffered TCM 199 (TCM 199B) with 5 mg/ml each of follicle stimulating hormone (FSH), lutenizing hormone (LH), 1 mg/ml of estradiol and 50 mg/ml of gentamycin sulfate. Control medium was additionally supplemented with 20% (v/v) BEF, OFF or ESS for IVM of oocytes. TCM 199B supplemented with FCS 10% (v/v), gentamycin sulfate 50 mg/ml, streptomycin 0.1 mg/ml and penicillin 100 IU/ml for culture of granulose cells. All media were sterilized by ®ltration through 0.22 mm ®lters (Sartorius, Germany) and equilibrated with 5% carbon dioxide in air in a humidi®ed atmosphere at 38.5 8C for at least 2 h. 2.3. Collection and processing of biological materials Sheep ovaries were collected from ewes of mixed breeds from a slaughter house and transported to the laboratory, a distance of about 200 km, within 5±6 h in warm (35 8C) phosphate buffered saline (PBS) supplemented with penicillin 1000 IU/ml and streptomy-
cin 1000 mg/ml. In the laboratory, the antral follicles (2±5 mm in diameter) on the ovaries were aspirated using a 22-gauge needle attached to a plastic 5 ml syringe containing 1 ml of oocyte handling medium. The cumulus oocyte complexes (COC) were isolated under a stereo zoom microscope (Nikon Corporation, Japan) and graded as good, fair or poor (Fig. 1A±C). Only good and fair oocytes were considered acceptable and used in IVM experiments. Follicular ¯uid was aspirated from transparent and healthy looking ovarian follicles greater than 4 mm in diameter. Pooled follicular ¯uid samples were centrifuged at 2000 rpm for 10 min. Supernatant was carefully removed and re-centrifuged as above. Finally the supernatant was ®ltered and stored at 20 8C in 1 ml aliquots until used. On the day of use, the follicular ¯uid was heated for 30 min at 56 8C in a water bath to inactivate the complement. Blood from jugular veins of ewes on the second day of estrus was collected in sterile glass tubes and was allowed to clot for 1±2 h at room temperature (25 8C). After the clot formed, the tubes were transferred to a refrigerator (4±5 8C) and the serum was allowed to separate. The serum was carefully collected and centrifuged at 1000 rpm for 10 min. The serum was sterilized by ®ltration and the complement was ®xed and stored until used. Follicles more than 4 mm in diameter were ¯ushed with 1 ml of Hanks balanced salt solution (HBSS) without calcium and magnesium using a 23-gauge needle attached to 2 ml plastic syringe. The aspirate was transferred to a test tube and 1 ml of hyaluronidase (150 IU/ml) was added to avoid coagulation. The contents were then centrifuged for 10 min at 2000 rpm. The supernatant was removed, the clot was re-suspended in TCM 199H and re-centrifuged. Finally the clot was diluted in 1 ml of granulosal cell culture medium. A 5 ml sample of this cell suspension was used to estimate the concentration and proportion of live and dead cells using Trypan blue stain following the procedure provided by the manufacturer. The cell concentration was adjusted to 2 106 cells/ml by adding required quantity of culture medium. To establish monolayers 500 ml of this suspension was placed in each of the four wells of a multidish (Nunclon, Denmark) and cultured in 5% carbon dioxide in air at 38.5 8C for 5±6 days. 0.25 ml of the culture medium was replaced with fresh medium every 48 h.
B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36
33
Fig. 1. In vitro maturation of sheep oocytes. (A) Freshly collected good oocyte; (B) freshly collected fair oocyte; (C) freshly collected poor oocyte; (D) in vitro matured oocyte exhibiting ®rst polar body; (E) germinal vesicle stage; (F) germinal vesicle break down; (G) metaphase I; (H) anaphase; (I) telophase; (J) metaphase II.
Light weight mineral oil equilibrated with the appropriate culture medium overnight in a humidi®ed atmosphere of 5% carbon dioxide in air at 38.5 8C was used to cover the micro-drops of oocyte cultures. Good and fair COC were washed twice in oocyte handling medium and an additional two times in the appropriate maturation medium. Each day, 5±10 COC collected were randomly allotted to each of the experimental groups. However, on certain days some of the experimental groups were not
allotted any oocytes (Table 1). For studying the in¯uence of various treatments, 5±10 COC were placed in 50 ml micro-drops of differently supplemented culture media including control or on monolayers of granulosal cells. Oocytes in micro-drops were covered with mineral oil and cultured for 24 h at 38.5 8C under humidi®ed atmosphere of 5% carbon dioxide in air. Oocytes on granulosal cell layers were similarly incubated but without mineral oil covering.
34
B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36
Table 1 In vitro maturation of sheep oocytes in different media during breeding seasona Culture medium
BEF ESS OFF GCC Control Fresh a
No. of oocytes stained (replicates)
No. of oocytes in different nuclear maturation stages (% of stained)
130 137 131 134 106 117
100 (77) 118 (86) 100 (76) 110 (82) 61 (58) NIL
(18) (19) (18) (17) (17) (15)
M II b c b b a
MI
A/T
GVBD
GV
16 (12) b 9 (7) a 12 (9) a 11 (8) a 14 (13) a NIL
7 7 13 10 12 2
1 1 1 1 3 22
6 2 5 2 16 93
(6) ab (6) ab (10) bc (8) ab (11) c (2) a
(0.8) a (0.7) a (0.8) a (0.8) a (3) b (19) c
(5) a (2) a (4) a (2) a (15) b (80) c
Values within a column with different letters differ at P < 0:01.
2.4. Evaluation of oocyte maturation At the end of IVM period oocytes from different groups were separately denuded of cumulus cells by repeatedly passing through a narrow bore glass pipette. Samples were washed in Hoechst 33342 ¯uorescent dye (5 mg/ml) and incubated in the dye for 15 min at 38.5 8C. Subsequently oocytes were examined on an inverted microscope (Leica, Germany) for extrusion of ®rst polar body (Fig. 1D) and under ¯uorescent light for nuclear maturation. Nuclear maturation stages were germinal vesicle (GV), germinal vesicle break down (GVBD), metaphase I, anaphase I, telophase I and metaphase II (Fig. 1E±J). 2.5. Statistical analysis The data on mean oocytes collected per ovary and the proportions of good, fair and poor oocytes (Table 2) were analyzed by normal deviate test. Rates of IVM were analyzed using a mathematical model that included the ®xed effect due to culture medium (BEF, ESS, OFF and control) and breeding season (breeding and non-breeding) and residual error.
Data on IVM of oocytes in different media during breeding season (experiment 2) was analyzed using a mathematical model that included ®xed effect due to culture medium (BEF, ESS, OFF, GCC, control and fresh) and residual error. Signi®cance of difference was determined by Duncan's multiple range test (Snedecor and Cochran, 1989). 3. Results A total of 1281 sheep ovaries were collected at the slaughter house which resulted in 1691 oocytes (Table 2) during the breeding and non-breeding seasons. There was no difference between seasons in the mean number of oocytes recovered per ovary. The proportions of oocytes in different quality groups were similar between the breeding and non-breeding seasons. The proportions of oocytes exhibiting ®rst polar body after IVM in three different media during the breeding and non-breeding seasons (BEF: 30:7 3:39 versus 30:5 1:89; ESS: 30:6 6:65 versus 37:7 5:95; OFF: 33:7 7:74 versus 28:4 4:25) were
Table 2 Collection of ovarian follicular oocytes in sheepa Source No. No. No. No. No. No.
of of of of of of a
ovaries oocytes oocytes/ovary good oocytes (%) fair oocytes (%) poor oocytes (%)
Breeding season
Non-breeding season
Total
824 1180 1.43 0.55 a 491 (42) b 404 (34) c 285 (24) d
457 511 1.12 0.45 a 200 (39) b 165 (32) c 146 (29) d
1281 1691 1.32 0.50 691 (41) 569 (34) 431 (26)
Values with same letters within a row do not differ at P < 0:05.
B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36 Table 3 In vitro maturation of sheep oocytes in different media during two different seasonsa Culture medium
BEF (20% (v/v)) ESS (20% (v/v)) OFF (20% (v/v)) Control
Percentage oocytes in metaphase II S.E. (replicates) Non-breeding season
Breeding season
59.3 69.3 56.7 45.4
63.4 3.27 a (8) 71.4 2.69 b (8) 59.2 2.88 a (8) 47.6 3.18 c (8)
3.03 2.94 2.18 3.01
a (8) b (8) a (8) c (8)
a Values with same letters within a row do not differ at P < 0:05, but values within a column with different letters differ at P < 0:01.
similar (P < 0:05). The interaction between season and the medium was also not signi®cant. The in¯uence of different media on proportion of oocytes maturing to M II stage was not signi®cantly different between the two seasons (Table 3) nor was the interaction between season and treatment. However, different media used had supported in vitro maturation at signi®cantly different rates (Table 3). Thus, ESS had supported the highest rate of IVM during both the seasons. BEF and OFF supported similar rates of IVM which were lower than that supported by ESS but higher than the control. It is interesting to note that among freshly collected COC, only 80% during the breeding season (Table 1) and 86% (data not shown) during non-breeding season were at the GV stage (P < 0:05). During the breeding season the proportions of oocytes exhibiting ®rst polar body after IVM in ESS and GCC were equal (38:8 2:98 versus 39:7 3:61) and signi®cantly higher than in all other groups (BEF: 28:6 2:12; OFF: 28:3 2:43; control: 25:6 2:85). Similarly ESS and GCC supported the highest rate of IVM to M II stage followed by BEF and OFF, and the control with the lowest rate (Table 1). Signi®cantly higher proportions of oocytes remained in GV and GVBD stages during maturation in control medium. 4. Discussion The mean rate of recovery of oocytes at 1.32 per ovary obtained in this study is lower than 4 oocytes per ovary reported by Wahid et al. (1992), 2.17 by Datta et al. (1993) and 2.75 by Das et al. (1996). However, in
35
these reports, the number of ovaries used were relatively small (44±140 versus 1281 in the present study). Further Wahid et al. (1992) also reported that only 1.5 oocytes per ovary were obtained when the aspiration time per ovary was reduced to 1.03 min. After transporting the ovaries from a slaughterhouse located 200 km away it was necessary to aspirate the oocytes in the shortest possible time before culture. This may have reduced the oocyte recovery rate. The number and quality of oocytes obtained (Table 2) does not support differential folliculogenic activity of the ovaries during the breeding and nonbreeding seasons. It is, however, possible that while ovarian follicular growth does not differ between seasons, the amount of estrogens produced by the ovarian follicles may differ between the seasons resulting in the observed differences (Kaushish, 1994) in occurrence of estrus. Estimation of estrogen content of sheep ovarian follicles during the breeding and non-breeding seasons could clarify this. Alternatively, antral follicles that appear on ovaries during the non-breeding season may not grow to ovulatory size to produce suf®cient estrogens. The ability of oocytes to undergo IVM as assessed by extrusion of ®rst polar body and development to M II (Table 3) did not differ signi®cantly between the seasons but was dependent on the medium. In a separate series of experiments during the breeding season, ESS and GCC supported the highest rate of IVM (Table 1). It has been reported that the addition of estrus cow serum to culture medium supported high rates of IVM of sheep oocytes (Yadav et al., 1997). Estrus goat serum (Mogas et al., 1995, 1997) and estrus buffalo serum (Madan et al., 1994) were reported earlier to support high rates of IVM of oocytes from respective species. BEF is tested as a commercial serum substitute to support IVM of sheep oocytes. In the present study, BEF supported IVM of oocytes to a lesser extent than ESS and GCC. Nevertheless, it was better than the control medium. This is in contrast to the observation on goat oocytes (Devaraju et al., 1997), where BEF and GCC supported similar rates of IVM. The ability of BEF to support IVM of sheep oocytes may vary with species. In the present study, sheep follicular ¯uid supported lower IVM rate than ESS and GCC. In the only earlier report on the use of sheep follicular ¯uid, Sun et al.
36
B.S. Rao et al. / Small Ruminant Research 43 (2002) 31±36
(1994) observed a higher rate of IVM with OFM from follicles 6 mm or more in diameter. Follicular ¯uid collected from small follicles was reported to support lower rates of IVM (Sun et al., 1994). The relatively smaller size of the follicles from which the OFF was collected in the present study may have contributed to lower frequency of IVM. The IVM rates of 58±86% in different media were lower compared to published reports (Brun et al., 1986; Salykbaev et al., 1986; Agrawal and Polge, 1988; Szollosi et al., 1988; Holm et al., 1991; Sun et al., 1994; Baldassarre et al., 1994; Byrd et al., 1997). However, in the present study, 80% of freshly collected oocytes were in GV stage (Table 1) suggesting approximately 20% of oocytes collected were abnormal and had no ability to undergo IVM. Thus, the IVM rate may be adjusted for a 20% reduction in oocytes used. On this basis the IVM rate in this experiment would be between 72 (control) and 100% (ESS) which is comparable to the reports from other laboratories. 5. Conclusions The follicular oocytes collected from south Indian sheep both during the breeding and non-breeding seasons were similar in their ability to undergo IVM. Furthermore, higher rates of in vitro maturation of sheep oocytes may be possible by supplementation of TCM 199B with hormones and estrus sheep serum or on monolayers of granulosa cell culture. Acknowledgements This work was supported by a research grant from the ANGR Agricultural University to V.H. Rao, Graduate studies scholarship of the Government of Andhra Pradesh, India to B.S. Rao and junior research fellowship the Department of Biotechnology, Government of India to R. Vagdevi. We are grateful to Drs. K. Rekkas and S. Belibasaki for critical review of the manuscript and constructive suggestions. References Agrawal, K.P., Polge, C., 1988. In vitro maturation of sheep oocytes. Indian J. Anim. Reprod. 9, 8±9.
Baldassarre, H., Castro, T.E., Furnus, C.C., de Matos, D.G., 1994. In vitro maturation and fertilisation of sheep oocytes collected by laparoscopic folliculocentesis. Theriogenology 41, 159. Brun, J., Iritani, A., Leidl, W., 1986. Fertilisation in vitro of ovine follicular oocytes with ejaculated spermatozoa capacitated in a chemically de®ned medium. Zuchtygiene 21, 59±63. Byrd, S.R., Flores-Foxworth, G., Applewhite, A.A., Westhusin, M.E., 1997. In vitro maturation of ovine oocytes in a portable incubator. Theriogenology 47, 857±864. Das, S.K., Chauhan, M.S., Palta, P., Katiyar, P.K., Madan, M.L., 1996. Replacement of fetal bovine serum and FSH with buffalo follicular ¯uid in in vitro maturation of buffalo oocytes. Theriogenology 45, 245. Datta, T.K., Goswami, S.L., Das, S.K., 1993. Comparative ef®ciency of three oocyte recovery methods from sheep ovaries. Indian J. Anim. Sci. 63, 1178±1179. Devaraju, B., Naidu, K.S., Amarnath, D., Rao, K.V., Rao, V.H., 1997. Comparison of the effect of a commercial serum substitute and goat follicular ¯uid on nuclear maturation of goat oocytes. Theriogenology 47, 189. Holm, P., Irvine, B., Armstrong, D.T., Seamark, R.F., 1991. In vitro production of sheep blastocysts from IVM oocytes using frozen semen and oviduct epithelial cell co-culture for IVF. Theriogenology 35, 214. Kaushish, S.K., 1994. Sheep Production in the Tropics and Subtropics. Scienti®c Publishers, Jodphur, India, pp. 26±28. Madan, M.L., Singla, S.K., Chauhan, M.B., Manik, R.S., 1994. In vitro production and transfer of embryos in buffaloes. Theriogenology 41, 139±143. Mogas, T., Izquierdo, M.D., Palomo, M.J., Aramio, M.T., 1995. Effect of hormones serum source and culture system on the IVM and IVF of prepubertal goat oocytes and subsequent embryo development. Theriogenology 43, 284. Mogas, T., Palomo, M.J., Iqzuieriz, M.D., Paramio, M.T., 1997. Developmental capacity of in vitro matured and fertilised oocytes from prepubertal and adult goats. Theriogenology 47, 1189±1203. Salykbaev, T., Dombroskii, N.N., Dzhienbaeva, R.S., Murazamadiev, A.M., 1986. Ability of oocytes from sheep of different ages to mature when incubated within the follicles. Eksperimential Noi Biologii Akademiya Nauk, Kazakhskoi SSR 19, 167±169. Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods. Iowa State University Press, Ames, Iowa, USA. Sun, F.J., Holm, P., Irvine, B., Seamark, R.F., 1994. Effect of sheep and human follicular ¯uid on the maturation of sheep oocytes in vitro. Theriogenology 41, 981±988. Szollosi, D., Deslmedi, V., Crozet, N., Drender, C., 1988. In vitro maturation of sheep ovarian oocytes. Reprod. Nutr. Develop. 13, 1047±1080. Wahid, H., Gordan, I., Sharif, H., Lonergan, P., Monaghan, P., Gallagher, M., 1992. Effect and ef®cacy of recovery methods for obtaining ovine follicular oocytes for in vitro procedures. Theriogenology 37, 318. Yadav, B.R., Katiyar, R.K., Chauhan, M.S., Madan, M.L., 1997. Chromosome con®guration during in vitro maturation of goat, sheep and buffalo oocytes. Theriogenology 47, 943±951.