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Peritoneal fluid from rabbits or goats as media for in vitro maturation, fertilization and initial culture of caprine oocytes
Animal Reproduction Science 54 Ž1999. 195–201
Peritoneal fluid from rabbits or goats as media for in vitro maturation, fertilization and initial culture of caprine oocytes Rakesh Kumar Malik a,) , Inder Singh Lohan a , Om Prakash Dhanda a , Om Kanwar Hooda b, Sajjan Singh a
b
Department of Animal Production Physiology, College of Animal Sciences, Chaudhary Charan Singh, Haryana Agricultural UniÕersity, Hisar-125 004, Haryana, India b Central Institute for Research on Buffaloes, Sirsa Road, Hisar-125 001, Haryana, India Accepted 21 May 1998
Abstract The efficacy of peritoneal fluid from rabbit and goat for in vitro maturation, fertilization and initial culture of embryos from caprine oocytes was evaluated. Peritoneal fluid was collected from adult female goats Ž n s 9. or rabbits Ž n s 9.. Good quality oocytes were subjected to in vitro maturation and fertilization in three different media viz. Tissue Culture Medium ŽTCM-199., goat Peritoneal Fluid ŽgPF. and rabbit Peritoneal fluid ŽrPF.. Maturation rates were 74.7 " 2.07% and 63.6 " 5.28% in TCM-199, gPF 65.8 " 2.54% and 55.6 " 3.79%, and rPF 57.7 " 1.78% and 44.6 " 3.01% when evaluated on the basis of cumulus cell expansion and the achievement of metaphase-II stage, respectively. However, no significant differences were observed in respect of maturation rate between the control and gPF and between gPF and rPF groups. Freshly ejaculated buck semen was treated with heparin Ž10 mgrml. and after 45 min incubation with heparin, 8.0% sperm were live and acrosome reacted. The proportions of fertilized oocytes based on male and female pronuclei formation or on cleavage development were 50.5 " 5.03, 42.3 " 3.15 and 34.2 " 1.98%; 31.0 " 2.80, 27.9 " 2.12 and 21.8 " 1.69% for TCM, gPF and rPF, respectively. It was concluded that peritoneal fluids either from goats or rabbits could be used as an alternative medium to TCM-199. However, further research is required to confirm its efficacy for embryo development up to the blastocyst stage. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Goat; Rabbit; Peritoneal fluid; In vitro maturation; IVF
)
Corresponding author. Tel.: q91-1662-31171-4235; fax: q91-1662-34952.
0378-4320r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 1 0 0 - 6
196
R.K. Malik et al.r Animal Reproduction Science 54 (1999) 195–201
1. Introduction TCM-199 supplemented with additives has been the most widely used medium for in vitro embryo production in farm species. However, it is expensive and not readily available in developing countries because it has to be imported thereby increasing the cost of embryo production. Storage of this medium in these circumstances is a further problem. Consequently, there is a need to search for alternative media. Several biological fluids such as egg saline medium ŽDowling, 1949.; serum ŽBrock and Rowson, 1952.; follicular fluid ŽSreenan et al., 1968. and rabbit peritoneal fluid ŽCollas et al., 1991; Chung et al., 1992., have been used with varying degrees of success for culture of oocytesrembryos of different species. The rationale for using rabbit peritoneal fluid for in vitro maturation, fertilization and co-culture has emanated from its already established ability to carry the conceptus to full term ŽBland-Sutton, 1904; Schumann, 1931; Buckley and Caine, 1979.. Recently, Collas et al. Ž1991., Chung et al. Ž1992. and Saini Ž1994. have reported maturation rates of 92 and 97% in rPF for bovine and caprine oocytes, respectively. Therefore, the efficiency of this fluid was compared with TCM-199 and peritoneal fluid from goats Žhomologous effect. in terms of their ability to support the maturation, fertilization and initial culture of goat oocytes.
2. Materials and methods 2.1. Goat and rabbit Peritoneal Fluid (gPF and rPF) collection The peritoneal fluid was aspirated using a 5 ml sterilized disposable syringe from adult female rabbits Ž n s 9. and goats Ž n s 9. after applying a local anaesthetic, Xylocaine 2% ŽLignocaine Hydrochloride ASTRA-IDL, Sweden. on the abdominal musculature at the site of needle puncture and pooled, separately, for each species. Strict haemostasis was maintained during aspiration to avoid contamination of fluid with blood. The gPF and rPF were centrifuged at 15 000 rpm for 1 min, the supernatant fraction of fluid filtered through a 0.22 mm millipore filter and antibiotic–antimycotic solution added Ž10 mlrml, A-7292; Sigma Chemical, USA.. From this fluid 100 ml drops were made in petri dishes ŽTarson, India., covered with light paraffin oil ŽSamir-Tech-Chem. Industry, Vadodara, India. and incubated overnight in 5% CO 2 at 38.58C. 2.2. Collection, recoÕery and selection of oocytes The study was conducted over a period of 7 months starting from September through March in two phases. In experiment 1, goat oocyte maturation was studied whilst, maturation, fertilization and early embryo culture was examined in experiment 2. In each experiment three maturationrculture media ŽTCM-199, goat peritoneal fluid and
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rabbit peritoneal fluid. were compared. Each treatment was replicated 13 times in each experiment. A total of 827 and 681 oocytes Ž320 and 264 ovaries. were utilized for experiments 1 and 2, respectively. One replicate was conducted per week commencing with the collection of ovaries which ranged between 15 to 30 on a single day Žcollection. from local abattoirs and transferred to the laboratory within 2–3 h in sterile physiological saline supplemented with antibiotics Ž200 IUrml benzyl penicilline and 200 mgrml streptomycin sulphate. in a thermos flask at 30–358C. The ovaries were rinsed three times with sterile saline, trimmed of excess tissue, immersed in 70% ethanol ŽBengal Chemicals, India. for 15–20 s and rinsed again three times in sterile saline. Visible antral follicles Ž) 1 mm. in diameter were punctured with a sterilized scalpel blade in TCM-199 ŽM-2520, Sigma. supplemented with 50 mgrml pyruvic acid ŽP-3662, Sigma.; 5.5 mgrml D-glucose ŽG-6152, Sigma.; 2.2 mgrml sodium bicarbonate ŽGR grade, Loba-Chemie, India.; 50 mgrml gentamicin sulfate ŽG-1264, Sigma.; 1% oestrous goat serum ŽOGS. and 1 IUrml heparin ŽSigma.. Oocytes with two or more layers of granulosa cells and homogenous cytoplasm were selected and washed twice in a 15 ml conical centrifuge tube ŽBorosil, India. in the TCM-199 medium; then washed four times in microdrops of this medium supplemented with 20% oestrus goat serum. These microdrops were prepared and kept in a CO 2 incubator 2–3 h before use. 2.3. Maturation, fertilization and initial culture of oocytes For maturation studies, duplicate 100 ml microdrops of each of the three maturation media were formed in 35 mm sterile petri dishes ŽTarson. providing two microdrops from each medium in three separate petri dishes per replicate and covered with sterile, CO 2 saturated, warm light paraffin oil. Groups of 10–12 oocytes were placed in each microdrop for 28 h at 38.58C and 5% CO 2 in air with 95% humidity. Maturation was determined by observing cumulus cell expansion and extrusion of the first polar bodyrmetaphase-II stage by fixation and staining as described by Malenko Ž1994.. The same maturation procedures were used in both experiments. For in vitro fertilization, freshly ejaculated spermatozoa from bucks of proven fertility were washed twice for 10 min at 400 g in modified-Defined Media Žm-DM. as described by Younis et al. Ž1991.. Highly motile sperm were separated by the swim-up method ŽParrish et al., 1988.. Capacitation was induced by incubating spermatozoa in m-DM supplemented with 10 mgrml heparin ŽH-3149, Sigma. and 388 mgrml caffeine–sodium–benzoate ŽC-4144, Sigma.. After 45 min of incubation at 38.58C in 5% CO 2 in air at high humidity, the acrosome reaction was confirmed by a dual stain procedure ŽDidion et al., 1989.. A total of 1–2 = 10 6 spermrml were added to 50 ml microdrops of fertilization medium, Fert-TALP ŽParrish et al., 1986. with five to seven matured oocytes were introduced per drop. The fertilization medium was supplemented with epinephrine and hypotaurine ŽMiller et al., 1992.. After 18 h co-incubation of sperm and oocytes, the oocytes were washed twice in 50 ml drops of TCM-199q 20% oestrous goat serum, gPF or rPF. Thereafter, four to five oocytes from each of the three media were fixed and stained with 1% aceto-orcein ŽMalenko, 1994.. The remaining oocytes were cultured for 48–72 h after insemination in the three culture media and subsequently evaluated for fertilization on the basis of cleavage Ž4–8 cell stage..
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2.4. Statistical design The data were analysed using non-parametric test of Mann–Whitney or Wilcoxon test using a statistical computer software package ‘SPSS version 6.0 for Windows’ in a personal computer. Probability of P - 0.05 was considered to be statistically significant.
3. Results On an average, 0.64 " 0.08 ml and 1.13 " 0.12 ml of peritoneal fluid was recovered from each goat and rabbit, respectively, with ranges of 0.3–1.2 ml and 0.6–2.0 ml. The mean total protein values for gPF and rPF were 30.8 " 4.80 mgrml and 24.7 " 1.90 mgrml, respectively. The in vitro maturation results for oocytes in different media in experiment 1 are presented in Table 1. The control medium ŽTCM-199. supported the highest maturation rate Ž74.7 " 2.07%. followed by gPF Ž65.8 " 2.54%. and rPF Ž57.7 " 1.78%., respectively, as judged by cumulus cell expansion. The maturation rate in control medium differed significantly Ž P - 0.05. from two other media which were also different from each other Ž P ) 0.05.. There was no significant difference in maturation rates between the control medium and gPF when assessed on a metaphase-II stage basis although the highest rate was in the control group Ž63.6 " 5.28%.. However, the maturation rates between control and rPF Ž44.6 " 3.01%. and between gPF Ž55.6 " 3.79%. and rPF groups did differ significantly Ž P - 0.05. when judged on a metaphase-II stage basis. In the test for the acrosome reaction of spermatozoa, 8.0% spermatozoa were live and acrosome reacted; 3.3% were dead and acrosome reacted; 75.2% were live with intact acrosome and 13.5% were dead with intact acrosome. The data for experiment 2, on in vitro fertilization and initial cleavage in the three media are presented in Table 2. The highest fertilization rates were observed in the control medium Ž50.5 " 5.03%. followed by gPF Ž42.3 " 3.15%. and rPF Ž34.2 " 1.98%., respectively, when assessed by pronuclei formation Ž18 h post sperm oocyte interaction.. The differences between control and gPF groups were not significant. Similarly, the fertilization rates for gPF and rPF did not differ significantly. However, the differences between control and rPF were significant Ž P - 0.05.. An identical
Table 1 Percent values ŽMean"S.E.. of caprine oocytes matured in vitro in different media Criteria of assessment of oocyte maturation
TCM-199q20% OGS Žcontrol.
gPF
rPF
Cumulus cell expansion Metaphase-II stage
74.7 a "2.07 63.6 a "5.28
65.8 b "2.54 55.6 a "3.79
57.7 c "1.78 44.6 b "3.01
gPF: Goat Peritoneal Fluid; OGS: Oestrous Goat Serum; rPF: Rabbit Peritoneal Fluid. Mean values Ž%. carrying dissimilar superscripts within a row differ from each other Ž P - 0.05..
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Table 2 Percent values ŽMean"S.E.. of caprine oocytes fertilized and cleaved in vitro in different media Criteria of assessment of oocyte fertilization
TCM-199q20% OGS ŽControl.
gPF
rPF
Male and female pronuclei formation Cleavage Ž4–8 cell stage.
50.5a "5.03 31.0 a "2.80
42.3 ab "3.15 27.9 a "2.12
34.2 b "1.98 21.8 b "1.69
gPF: Goat Peritoneal Fluid; OGS: Oestrous Goat Serum; rPF: Rabbit Peritoneal Fluid. Mean values Ž%. carrying dissimilar superscripts within a row differ from each other Ž P - 0.05..
pattern of treatment differences was observed when fertilization rate was assessed by cleavage and development to the 4–8 cell stage.
4. Discussion Surprisingly, rabbits yielded more peritoneal fluid than goats possibly because the peritoneal cavity of ruminants is anatomically ‘walled off’ by the fold of omentum ŽBhokre, 1993., or because the peritoneal fluid is distributed over a large surface area in goats. It was observed that the largest amount of fluid could be harvested from the site of the left lateral abdomen in-between the xiphoid sternum and the umbilicus in both goats and rabbits. The oocyte maturation rates observed for TCM-199 in this study were similar to those reported in goats by Song and Iritani Ž1988., Zhiming et al. Ž1990., Ling et al. Ž1992. and Mogas et al. Ž1995.. Younis et al. Ž1992., Smedt et al. Ž1992., Martino et al. Ž1994b., Pawshe et al. Ž1994. have reported higher maturation rates in this species ranging from 72 to 100%. A number of factors including recovery and processing of ovaries ŽXu et al., 1987., high plane of nutrition ŽYounis et al., 1991. and the priming of ovaries with hormones ŽSmedt et al., 1992. have been associated with higher maturation rates. There is evidence that many of the oocytes recovered from 1–2 mm follicles Žas was the case in the present study. do not reach Metaphase-II stage and remain arrested at Metaphase-I ŽSmedt et al., 1992; Martino et al., 1994a.. Saini Ž1994. has reported a very high maturation rate Ž92.22 " 1.26%. in rPF and similar observations have been made by Singh and Dhanda Ž1997., in a study of maturation of buffalo oocytes in rPF. There is an absence of published information relating to goat peritoneal fluid. The success of the peritoneal fluids in supporting oocyte maturation in this study may have been due to their total protein contents being similar to those of uterine and oviductal fluids ŽIritani et al., 1971. which seem to be essential for maturation and fertilization under in vivo conditions. The fertilization rates observed in the control medium ŽTCM-199. on the basis of male and female pronuclei formation in our study are in agreement with the findings of Younis et al. Ž1991., Martino et al. Ž1993., Mogas et al. Ž1995. and Palomo et al. Ž1995.. On a cleavage rate basis, the fertilization rates in this medium are comparable to the findings of Palomo et al. Ž1995.. Smedt et al. Ž1992., Keskintepe et al. Ž1994. and Mogas et al. Ž1995. have observed higher cleavage rates which might be due to the
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better quality of oocytes used for maturation which were recovered from large follicles Ž) 2 mm in diameter.. Goat peritoneal fluid appeared to be as effective as the control medium for in vitro maturation when assessed by metaphase-II stage data in experiment 1. The maturation rate from rPF group in this experiment was significantly lower Ž P - 0.05. to those obtained in control and gPF groups. Similar results, in terms of cleavage rate, were observed in experiment 2. It is concluded that goat peritoneal fluid could be used as an alternative medium to the conventional TCM-199 for generating goat embryos by in vitro techniques. Further research is required to improve cleavage rates and examine subsequent development to blastocyst stage embryos.
Acknowledgements The authors would like to thank the Head, Department of Animal Production Physiology, College of Animal Sciences, Chaudhary Charan Singh Haryana Agricultural University, Hisar ŽIndia. for providing the necessary facilities for conducting the present study.
References Bhokre, A.P., 1993. Peritoneum. In: Tyagi, R.P.S., Singh, J. ŽEds.., A Text Book of the Surgical Diseases of Cattle, Buffaloes, Camels, Sheep and Goats. CBS Publishers, Delhi, pp. 180–182. Bland-Sutton, J., 1904. Abdominal pregnancy in woman, cats, dogs and rabbits. Lancet 2, 1625–1627. Brock, H., Rowson, L.E., 1952. The production of viable bovine ova. J. Agric. Sci. 42, 479, Abstract. Buckley, P., Caine, A., 1979. A high incidence of abdominal pregnancy in Djungarian hamster. J. Reprod. Fert. 56, 679–682. Chung, K.S., Lee, H.T., Chung, H.M., Park, S.P., Oh, J.H., 1992. The effect of rabbit peritoneal fluid on in vitro maturation of bovine and porcine follicular oocytes. Proc. of VI AAAP Anim. Sci. Congr., Ahat, Bangkok, Abstract, p. 29. Collas, P., Duby, R.T., Robi, J.M., 1991. In vitro development of rabbit pronuclear embryos in rabbit peritoneal fluid. Biol. Reprod. 44, 1100–1107. Didion, B.A., Dobringsky, J.R., Giles, J.R., Graves, C.N., 1989. Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species. Gamete Res. 22, 51–57. Dowling, D.F., 1949. Problems of transportation of fertilized ova. J. Agric. Sci. 39, 374, Abstract. Iritani, A., Nishikawa, Y., Gomes, W.R., Vandemark, N.L., 1971. Secretion rates and chemical composition of oviduct and uterine fluids in rabbits. J. Anim. Sci. 33, 829–835. Keskintepe, L., Darwish, G.M., Kenimer, A.T., Brackett, B.G., 1994. Term development of caprine embryos derived from immature oocytes in vitro. Theriogenology 42, 527–535. Ling, L., Jufen, Q., Young, Z., 1992. In vitro fertilization of goat ovarian oocytes. Theriogenology 37, 247, Abstract. Malenko, G.P., 1994. An improved method for preparing whole specimens from bovine preimplantation embryos: a technique note. Theriogenology 41, 1207–1210. Martino, A., Mogas, T., Palomo, M.J., Paramio, M.T., 1993. Effect of oocyte and granulosa cell source used during in vitro maturation on in vitro fertilization of goat oocytes. Theriogenology 39, 265, Abstract. Martino, A., Mogas, T., Palomo, M.J., Paramio, M.T., 1994a. Meiotic competence of prepubertal goat oocytes. Theriogenology 41, 969–980.
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Martino, A., Palomo, M.J., Mogas, T., Paramio, M.T., 1994b. Influence of the collection technique of prepubertal goat oocytes on in vitro maturation and fertilization. Theriogenology 42, 859–873. Miller, G.F., Gleidt, D.L., Lester, T.D., Pierson, J.N., Rakes, J.M., Rorie, R.W., 1992. Addition of bovine oviductal epithelial cells ŽBOEC. andror penicillamine, hypotaurine and epinephrine ŽPHE. to bovine in vitro fertilization ŽIVF. medium increases the subsequent embryo cleavage rate. Theriogenology 37, 259, Abstract. Mogas, T., Izquierdo, M.D., Palomo, M.J., Paramio, 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, Abstract. Palomo, M.J., Mogas, T., Izquierdo, M.D., Paramio, M.T., 1995. Effect of heparin and sperm concentration on IVF of prepubertal goat oocytes. Theriogenology 43, 292, Abstract. Parrish, J.J., Susko-Parrish, J.L., Leibfried-Rutledge, M.L., Crister, E.S., Eyestone, W.H., First, N.L., 1986. Bovine in vitro fertilization with frozen thawed semen. Theriogenology 25, 591–600. Parrish, J.J., Susko-Parrish, J.L., Winek, M.A., First, N.L., 1988. Capacitation of bovine sperm by heparin. Biol. Reprod. 38, 1171–1180. Pawshe, C.H., Apparao, K.B.C., Jain, S.K., Totey, S.M., 1994. Biochemical studies of goat oocytes: timing of nuclear progression, effect of protein inhibitor and pattern of polypeptide synthesis during in vitro maturation. Theriogenology 42, 307–320. Saini, M.S., 1994. Studies on in vitro maturation, fertilization and co-culture of caprine oocytes. PhD Thesis submitted to Chaudhary Charan Singh, Haryana Agricultural University, Hisar, India. Schumann, E.A., 1931. Extra uterine pregnancy. Gynaecol. Obst. Monographs, Vol. VII-D. Appleton and Company, New York. Singh, S., Dhanda, O.P., 1997. Rabbit peritoneal fluid as a medium for in vitro maturation, fertilization and co-culture of buffalo oocytes. Proceedings Vth World Buffalo Congress, Caserta, Italy, Oct. 13–16, pp. 800–804. Smedt, V.De., Crozet, H., Ahmad-Ali, M., Martino, A., Cognie, Y., 1992. In vitro maturation and fertilization of goat oocytes. Theriogenology 37, 1049–1060. Song, H.B., Iritani, A., 1988. In vitro fertilization of goat follicular oocytes with epididymal spermatozoa. Korean J. Anim. Sci. 30, 636–642. Sreenan, J., Scanlon, P., Gordon, I., 1968. Culture of fertilized cow eggs. J. Agric. Sci. 70, 183, Abstract. Xu, K.P., Greve, T., Callesen, H., Hyttel, P., 1987. Pregnancy resulting from cattle oocytes matured and fertilized in vitro. J. Reprod. Fert. 81, 501–504. Younis, A.I., Zuelke, K.A., Harper, K.M., Oliveira, M.A.L., Brackett, B.G., 1991. In vitro fertilization of goat oocytes. Biol. Reprod. 44, 1177–1182. Younis, A.I., Keskintepe, L., Mackie, K., Brackett, B.G., 1992. In vitro maturation and fertilization of Toggenberg goat oocytes. Theriogenology 37, 330, Abstract. Zhiming, H., Jianchen, W., Jufen, Q., 1990. In vitro maturation of follicular oocytes from the mouse and dairy goat. Theriogenology 33, 364, Abstract.
Peritoneal fluid from rabbits or goats as media for in vitro maturation, fertilization and initial culture of caprine oocytes Rakesh Kumar Malik a,) , Inder Singh Lohan a , Om Prakash Dhanda a , Om Kanwar Hooda b, Sajjan Singh a
b
Department of Animal Production Physiology, College of Animal Sciences, Chaudhary Charan Singh, Haryana Agricultural UniÕersity, Hisar-125 004, Haryana, India b Central Institute for Research on Buffaloes, Sirsa Road, Hisar-125 001, Haryana, India Accepted 21 May 1998
Abstract The efficacy of peritoneal fluid from rabbit and goat for in vitro maturation, fertilization and initial culture of embryos from caprine oocytes was evaluated. Peritoneal fluid was collected from adult female goats Ž n s 9. or rabbits Ž n s 9.. Good quality oocytes were subjected to in vitro maturation and fertilization in three different media viz. Tissue Culture Medium ŽTCM-199., goat Peritoneal Fluid ŽgPF. and rabbit Peritoneal fluid ŽrPF.. Maturation rates were 74.7 " 2.07% and 63.6 " 5.28% in TCM-199, gPF 65.8 " 2.54% and 55.6 " 3.79%, and rPF 57.7 " 1.78% and 44.6 " 3.01% when evaluated on the basis of cumulus cell expansion and the achievement of metaphase-II stage, respectively. However, no significant differences were observed in respect of maturation rate between the control and gPF and between gPF and rPF groups. Freshly ejaculated buck semen was treated with heparin Ž10 mgrml. and after 45 min incubation with heparin, 8.0% sperm were live and acrosome reacted. The proportions of fertilized oocytes based on male and female pronuclei formation or on cleavage development were 50.5 " 5.03, 42.3 " 3.15 and 34.2 " 1.98%; 31.0 " 2.80, 27.9 " 2.12 and 21.8 " 1.69% for TCM, gPF and rPF, respectively. It was concluded that peritoneal fluids either from goats or rabbits could be used as an alternative medium to TCM-199. However, further research is required to confirm its efficacy for embryo development up to the blastocyst stage. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Goat; Rabbit; Peritoneal fluid; In vitro maturation; IVF
)
Corresponding author. Tel.: q91-1662-31171-4235; fax: q91-1662-34952.
0378-4320r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 1 0 0 - 6
196
R.K. Malik et al.r Animal Reproduction Science 54 (1999) 195–201
1. Introduction TCM-199 supplemented with additives has been the most widely used medium for in vitro embryo production in farm species. However, it is expensive and not readily available in developing countries because it has to be imported thereby increasing the cost of embryo production. Storage of this medium in these circumstances is a further problem. Consequently, there is a need to search for alternative media. Several biological fluids such as egg saline medium ŽDowling, 1949.; serum ŽBrock and Rowson, 1952.; follicular fluid ŽSreenan et al., 1968. and rabbit peritoneal fluid ŽCollas et al., 1991; Chung et al., 1992., have been used with varying degrees of success for culture of oocytesrembryos of different species. The rationale for using rabbit peritoneal fluid for in vitro maturation, fertilization and co-culture has emanated from its already established ability to carry the conceptus to full term ŽBland-Sutton, 1904; Schumann, 1931; Buckley and Caine, 1979.. Recently, Collas et al. Ž1991., Chung et al. Ž1992. and Saini Ž1994. have reported maturation rates of 92 and 97% in rPF for bovine and caprine oocytes, respectively. Therefore, the efficiency of this fluid was compared with TCM-199 and peritoneal fluid from goats Žhomologous effect. in terms of their ability to support the maturation, fertilization and initial culture of goat oocytes.
2. Materials and methods 2.1. Goat and rabbit Peritoneal Fluid (gPF and rPF) collection The peritoneal fluid was aspirated using a 5 ml sterilized disposable syringe from adult female rabbits Ž n s 9. and goats Ž n s 9. after applying a local anaesthetic, Xylocaine 2% ŽLignocaine Hydrochloride ASTRA-IDL, Sweden. on the abdominal musculature at the site of needle puncture and pooled, separately, for each species. Strict haemostasis was maintained during aspiration to avoid contamination of fluid with blood. The gPF and rPF were centrifuged at 15 000 rpm for 1 min, the supernatant fraction of fluid filtered through a 0.22 mm millipore filter and antibiotic–antimycotic solution added Ž10 mlrml, A-7292; Sigma Chemical, USA.. From this fluid 100 ml drops were made in petri dishes ŽTarson, India., covered with light paraffin oil ŽSamir-Tech-Chem. Industry, Vadodara, India. and incubated overnight in 5% CO 2 at 38.58C. 2.2. Collection, recoÕery and selection of oocytes The study was conducted over a period of 7 months starting from September through March in two phases. In experiment 1, goat oocyte maturation was studied whilst, maturation, fertilization and early embryo culture was examined in experiment 2. In each experiment three maturationrculture media ŽTCM-199, goat peritoneal fluid and
R.K. Malik et al.r Animal Reproduction Science 54 (1999) 195–201
197
rabbit peritoneal fluid. were compared. Each treatment was replicated 13 times in each experiment. A total of 827 and 681 oocytes Ž320 and 264 ovaries. were utilized for experiments 1 and 2, respectively. One replicate was conducted per week commencing with the collection of ovaries which ranged between 15 to 30 on a single day Žcollection. from local abattoirs and transferred to the laboratory within 2–3 h in sterile physiological saline supplemented with antibiotics Ž200 IUrml benzyl penicilline and 200 mgrml streptomycin sulphate. in a thermos flask at 30–358C. The ovaries were rinsed three times with sterile saline, trimmed of excess tissue, immersed in 70% ethanol ŽBengal Chemicals, India. for 15–20 s and rinsed again three times in sterile saline. Visible antral follicles Ž) 1 mm. in diameter were punctured with a sterilized scalpel blade in TCM-199 ŽM-2520, Sigma. supplemented with 50 mgrml pyruvic acid ŽP-3662, Sigma.; 5.5 mgrml D-glucose ŽG-6152, Sigma.; 2.2 mgrml sodium bicarbonate ŽGR grade, Loba-Chemie, India.; 50 mgrml gentamicin sulfate ŽG-1264, Sigma.; 1% oestrous goat serum ŽOGS. and 1 IUrml heparin ŽSigma.. Oocytes with two or more layers of granulosa cells and homogenous cytoplasm were selected and washed twice in a 15 ml conical centrifuge tube ŽBorosil, India. in the TCM-199 medium; then washed four times in microdrops of this medium supplemented with 20% oestrus goat serum. These microdrops were prepared and kept in a CO 2 incubator 2–3 h before use. 2.3. Maturation, fertilization and initial culture of oocytes For maturation studies, duplicate 100 ml microdrops of each of the three maturation media were formed in 35 mm sterile petri dishes ŽTarson. providing two microdrops from each medium in three separate petri dishes per replicate and covered with sterile, CO 2 saturated, warm light paraffin oil. Groups of 10–12 oocytes were placed in each microdrop for 28 h at 38.58C and 5% CO 2 in air with 95% humidity. Maturation was determined by observing cumulus cell expansion and extrusion of the first polar bodyrmetaphase-II stage by fixation and staining as described by Malenko Ž1994.. The same maturation procedures were used in both experiments. For in vitro fertilization, freshly ejaculated spermatozoa from bucks of proven fertility were washed twice for 10 min at 400 g in modified-Defined Media Žm-DM. as described by Younis et al. Ž1991.. Highly motile sperm were separated by the swim-up method ŽParrish et al., 1988.. Capacitation was induced by incubating spermatozoa in m-DM supplemented with 10 mgrml heparin ŽH-3149, Sigma. and 388 mgrml caffeine–sodium–benzoate ŽC-4144, Sigma.. After 45 min of incubation at 38.58C in 5% CO 2 in air at high humidity, the acrosome reaction was confirmed by a dual stain procedure ŽDidion et al., 1989.. A total of 1–2 = 10 6 spermrml were added to 50 ml microdrops of fertilization medium, Fert-TALP ŽParrish et al., 1986. with five to seven matured oocytes were introduced per drop. The fertilization medium was supplemented with epinephrine and hypotaurine ŽMiller et al., 1992.. After 18 h co-incubation of sperm and oocytes, the oocytes were washed twice in 50 ml drops of TCM-199q 20% oestrous goat serum, gPF or rPF. Thereafter, four to five oocytes from each of the three media were fixed and stained with 1% aceto-orcein ŽMalenko, 1994.. The remaining oocytes were cultured for 48–72 h after insemination in the three culture media and subsequently evaluated for fertilization on the basis of cleavage Ž4–8 cell stage..
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2.4. Statistical design The data were analysed using non-parametric test of Mann–Whitney or Wilcoxon test using a statistical computer software package ‘SPSS version 6.0 for Windows’ in a personal computer. Probability of P - 0.05 was considered to be statistically significant.
3. Results On an average, 0.64 " 0.08 ml and 1.13 " 0.12 ml of peritoneal fluid was recovered from each goat and rabbit, respectively, with ranges of 0.3–1.2 ml and 0.6–2.0 ml. The mean total protein values for gPF and rPF were 30.8 " 4.80 mgrml and 24.7 " 1.90 mgrml, respectively. The in vitro maturation results for oocytes in different media in experiment 1 are presented in Table 1. The control medium ŽTCM-199. supported the highest maturation rate Ž74.7 " 2.07%. followed by gPF Ž65.8 " 2.54%. and rPF Ž57.7 " 1.78%., respectively, as judged by cumulus cell expansion. The maturation rate in control medium differed significantly Ž P - 0.05. from two other media which were also different from each other Ž P ) 0.05.. There was no significant difference in maturation rates between the control medium and gPF when assessed on a metaphase-II stage basis although the highest rate was in the control group Ž63.6 " 5.28%.. However, the maturation rates between control and rPF Ž44.6 " 3.01%. and between gPF Ž55.6 " 3.79%. and rPF groups did differ significantly Ž P - 0.05. when judged on a metaphase-II stage basis. In the test for the acrosome reaction of spermatozoa, 8.0% spermatozoa were live and acrosome reacted; 3.3% were dead and acrosome reacted; 75.2% were live with intact acrosome and 13.5% were dead with intact acrosome. The data for experiment 2, on in vitro fertilization and initial cleavage in the three media are presented in Table 2. The highest fertilization rates were observed in the control medium Ž50.5 " 5.03%. followed by gPF Ž42.3 " 3.15%. and rPF Ž34.2 " 1.98%., respectively, when assessed by pronuclei formation Ž18 h post sperm oocyte interaction.. The differences between control and gPF groups were not significant. Similarly, the fertilization rates for gPF and rPF did not differ significantly. However, the differences between control and rPF were significant Ž P - 0.05.. An identical
Table 1 Percent values ŽMean"S.E.. of caprine oocytes matured in vitro in different media Criteria of assessment of oocyte maturation
TCM-199q20% OGS Žcontrol.
gPF
rPF
Cumulus cell expansion Metaphase-II stage
74.7 a "2.07 63.6 a "5.28
65.8 b "2.54 55.6 a "3.79
57.7 c "1.78 44.6 b "3.01
gPF: Goat Peritoneal Fluid; OGS: Oestrous Goat Serum; rPF: Rabbit Peritoneal Fluid. Mean values Ž%. carrying dissimilar superscripts within a row differ from each other Ž P - 0.05..
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Table 2 Percent values ŽMean"S.E.. of caprine oocytes fertilized and cleaved in vitro in different media Criteria of assessment of oocyte fertilization
TCM-199q20% OGS ŽControl.
gPF
rPF
Male and female pronuclei formation Cleavage Ž4–8 cell stage.
50.5a "5.03 31.0 a "2.80
42.3 ab "3.15 27.9 a "2.12
34.2 b "1.98 21.8 b "1.69
gPF: Goat Peritoneal Fluid; OGS: Oestrous Goat Serum; rPF: Rabbit Peritoneal Fluid. Mean values Ž%. carrying dissimilar superscripts within a row differ from each other Ž P - 0.05..
pattern of treatment differences was observed when fertilization rate was assessed by cleavage and development to the 4–8 cell stage.
4. Discussion Surprisingly, rabbits yielded more peritoneal fluid than goats possibly because the peritoneal cavity of ruminants is anatomically ‘walled off’ by the fold of omentum ŽBhokre, 1993., or because the peritoneal fluid is distributed over a large surface area in goats. It was observed that the largest amount of fluid could be harvested from the site of the left lateral abdomen in-between the xiphoid sternum and the umbilicus in both goats and rabbits. The oocyte maturation rates observed for TCM-199 in this study were similar to those reported in goats by Song and Iritani Ž1988., Zhiming et al. Ž1990., Ling et al. Ž1992. and Mogas et al. Ž1995.. Younis et al. Ž1992., Smedt et al. Ž1992., Martino et al. Ž1994b., Pawshe et al. Ž1994. have reported higher maturation rates in this species ranging from 72 to 100%. A number of factors including recovery and processing of ovaries ŽXu et al., 1987., high plane of nutrition ŽYounis et al., 1991. and the priming of ovaries with hormones ŽSmedt et al., 1992. have been associated with higher maturation rates. There is evidence that many of the oocytes recovered from 1–2 mm follicles Žas was the case in the present study. do not reach Metaphase-II stage and remain arrested at Metaphase-I ŽSmedt et al., 1992; Martino et al., 1994a.. Saini Ž1994. has reported a very high maturation rate Ž92.22 " 1.26%. in rPF and similar observations have been made by Singh and Dhanda Ž1997., in a study of maturation of buffalo oocytes in rPF. There is an absence of published information relating to goat peritoneal fluid. The success of the peritoneal fluids in supporting oocyte maturation in this study may have been due to their total protein contents being similar to those of uterine and oviductal fluids ŽIritani et al., 1971. which seem to be essential for maturation and fertilization under in vivo conditions. The fertilization rates observed in the control medium ŽTCM-199. on the basis of male and female pronuclei formation in our study are in agreement with the findings of Younis et al. Ž1991., Martino et al. Ž1993., Mogas et al. Ž1995. and Palomo et al. Ž1995.. On a cleavage rate basis, the fertilization rates in this medium are comparable to the findings of Palomo et al. Ž1995.. Smedt et al. Ž1992., Keskintepe et al. Ž1994. and Mogas et al. Ž1995. have observed higher cleavage rates which might be due to the
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better quality of oocytes used for maturation which were recovered from large follicles Ž) 2 mm in diameter.. Goat peritoneal fluid appeared to be as effective as the control medium for in vitro maturation when assessed by metaphase-II stage data in experiment 1. The maturation rate from rPF group in this experiment was significantly lower Ž P - 0.05. to those obtained in control and gPF groups. Similar results, in terms of cleavage rate, were observed in experiment 2. It is concluded that goat peritoneal fluid could be used as an alternative medium to the conventional TCM-199 for generating goat embryos by in vitro techniques. Further research is required to improve cleavage rates and examine subsequent development to blastocyst stage embryos.
Acknowledgements The authors would like to thank the Head, Department of Animal Production Physiology, College of Animal Sciences, Chaudhary Charan Singh Haryana Agricultural University, Hisar ŽIndia. for providing the necessary facilities for conducting the present study.
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