- Email: [email protected]
Effects of resazurin on bovine oocyte fertilization and embryo development in vitro
Animal Reproduction Science 51 Ž1998. 205–213
Effects of resazurin on bovine oocyte fertilization and embryo development in vitro S. Wang a
a,)
, G.R. Holyoak a , G. Liu a,b, T.J. Bunch c , R.C. Evans a , T.D. Bunch a
Department of Animal, Dairy and Veterinary Sciences, Utah State UniÕersity, Logan, UT 84322-9400, USA b Department of Biotechnology, Northeast Agricultural UniÕersity, Harbin 150030, China c UniÕersity of Utah School of Medical, Salt Lake City, UT 84113, USA Accepted 20 February 1998
Abstract Resazurin is a redox dye Ž7-hydroxy-3H-phenoxazin-3-one-10-oxide. used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation ŽIVM., fertilization ŽIVF. and culture ŽIVC.. In Experiment 1, bovine oocytes Ž n s 2767. were fertilized with spermatozoa exposed to resazurin Ž17.6 m grml. for 0, 15, 30, 60 min, respectively. There was no significant Ž P ) 0.05. difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes Ž n s 1671. were treated with resazurin Ž1.8 m grml. during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant Ž P ) 0.05. difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytesrembryos were treated with resazurin during IVC or IVMrIVFrIVC was significantly Ž P - 0.05. less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with
)
Corresponding author: Tel.: q1 435 797 3970; fax: q1 435 797 3494; e-mail: [email protected]
0378-4320r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 0 7 2 - 4
206
S. Wang et al.r Animal Reproduction Science 51 (1998) 205–213
resazurin may be detrimental to embryonic development. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cattle—reproductive technology; Resazurin; Oocyte maturation; In vitro fertilization; Embryo culture
1. Introduction Resazurin is a redox dye Ž7-hydroxy-3H-phenoxazin-3-one-10-oxide. traditionally used for assessing microbiological quality of milk and dairy products ŽMattila-Sandholm et al., 1991.. Because the reduction of resazurin is characterized by color changes, and because the intermediate of the resazurin reduction reaction is a strong fluorogenic redox indicator Žresorufin., minor changes in concentration of resazurin can be detected by photometric or fluorometric methods ŽMattila-Sandholm et al., 1991.. This chemical property of resazurin dye allows for potential uses to quantitatively study the functional status of eukaryotic cells ŽFang et al., 1997.. Resazurin has been used to measure the metabolic activity of spermatozoa and to assess fertilizing capacity ŽErb and Ehler, 1952; Glass et al., 1991; Dart et al., 1994; Carter et al., 1995.. When activated by metabolically viable spermatozoa, resazurin is first reduced to resorufin and then to dihydroresorufin. The reduction reaction is manifested by visual changes in dye color from blue Žresazurin. to pink Žresorufin. and further to white Ždihydroresorufin. and is measurable quantitatively by spectrophotometry ŽWang et al., 1996.. The rate of color change associates to the potential fertilizing capacity of spermatozoa. Usually, the faster the color change from blue to pink and to white, the better the semen quality ŽGlass et al., 1991; Dart et al., 1994.. The resazurin sperm assay mainly depends on the concentration of live sperm ŽMahmoud et al., 1994; Zalata et al., 1995.. As a test for fertility, the assay correlates better than most sperm characteristics commonly used in routine semen analysis ŽComhaire et al., 1987; Fuse et al., 1993; Mahmoud et al., 1994; Zalata et al., 1995.. Resazurin is also a more reliable measurement of metabolic activity for sperm quality than ATP measurement ŽHinting et al., 1988; Comhaire and Vermeulen, 1995.. The simplicity and accuracy of the resazurin assay has been supported by the data from fertility tests in humans ŽGlass et al., 1991. and several domestic animals ŽDart et al., 1994; Comhaire and Vermeulen, 1995; Carter et al., 1995; Cooper et al., 1995.. Due to its sensitivity and reproductivity as an indicator of dehydrogenase activity in the metabolic assessment of cell viability ŽMattila-Sandholm et al., 1991., resazurin has the potential to be used in non-invasive assessments of semen quality and embryo viability. However, there is a lack of information on the effects of resazurin on fertilization and the effects on embryo development. Recent advances in in vitro maturation ŽIVM., in vitro fertilization ŽIVF. and in vitro embryo culture ŽIVC. provide an analytical methodology for assessing fertilizing capacity of spermatozoa and the developmental potential of preimplantation embryos ŽBavister, 1995.. The effects of resazurin can, therefore, be evaluated by the use of in vitro models. The objectives of this study were Ž1. to examine the effects of resazurin treatment on fertilizing capacity of spermatozoa using the technique of in vitro production of bovine
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embryos and Ž2. to investigate the effects of resazurin on the development of bovine oocytesrembryos during oocyte maturation, oocyte fertilization and embryo development in vitro.
2. Materials and methods Ovaries were collected from a local abattoir. Oocytes were aspirated from small antral follicles Ž3–8 mm in diameter. as described by Hawk and Wall Ž1994.. Cumulus oocyte complexes ŽCOCs. with evenly granulated ooplasm and surrounded by several layers Žat least 3 to 4 layers. of compact cumulus cells were selected for use according to the oocyte grading system described by Hawk and Wall Ž1994.. Oocytes were washed three times with HEPES-TALP solution ŽParrish et al., 1988. and once with maturation medium. The maturation medium consisted of M-199 plus 10% Žvolrvol. fetal bovine serum ŽFBS., 25 mM HEPES, 2 mM glutamine, 0.25 mM sodium pyruvate, 0.5 m grml ovine FSH ŽF-4520, Sigma, St. Louis, MO, USA., 5.0 m grml ovine LH ŽL-5269, Sigma. 1.0 m grml estradiol ŽE-2258, Sigma.. In vitro maturation ŽIVM. of oocytes followed the procedure of Sirard et al. Ž1988.. Polystyrene plastic 4-well culture petri dishes ŽNunclon w , Nunc, Naperville, IL, USA. were used for IVM culture. Each well contained 500 m l IVM medium covered with paraffin oil. Approximately 40 to 65 oocytes were transferred to the IVM medium and cultured in a humidified 5% CO 2 atmosphere at 398C for 24 h. Cryopreserved bovine semen was used for in vitro fertilization ŽIVF.. Live sperm were separated by Percoll gradients Ž45% and 90% on the upper and lower layers, respectively. and centrifuged at 500 = g for 30 min. Motile spermatozoa were added to the fertilization medium ŽFert-TALP, Parrish et al., 1988. to provide a final concentration of 2 = 10 6 per ml. Capacitation of spermatozoa occurred in Fert-TALP containing 10 m g heparinrml and 0.6% fatty acid free bovine serum albumin. IVM matured oocytes were added to Fert-TALP containing spermatozoa and cultured in plastic 4-well petri dishes ŽNunclon w , Nunc. under paraffin oil in a humidified 5% CO 2 atmosphere at 398C for 17 h. Each well contained 500 m l Fert-TALP and approximately 40 to 65 oocytes. Cumulus and corona cells were removed from ova by vortexing in HEPES-TALP supplemented with 0.3% bovine serum albumin for 3 min. The presumptive zygotes were then cultured in plastic 4-well petri dishes ŽNunclon w , Nunc. under paraffin oil at 398C in a humidified 5% CO 2 atmosphere. A modified CR2 medium ŽWang et al., 1997. comprising 108.3 mM NaCl, 2.9 mM KCl, 24.9 mM NaHCO 3 , 2.5 mM hemicalcium lactate, 0.5 mM sodium pyruvate, BME amino acids ŽB-6766, Sigma., MEM non-essential amino acids ŽM-7145, Sigma., 0.5 mM glycine, 0.5 mM alanine, 1.0 mM glutamine, 1.0 mM glucose, and antibiotics was used to culture embryos. Each well contained 500 m l CR2 medium with approximately 40 to 60 oocytes. During culture, medium was changed every other day so that it was supplemented with 5% Žvolrvol. fetal bovine serum on Day 1 ŽDay 0 s IVF., 10% on Day 3, 15% on Day 5 and 20% on Day 7 of culture ŽZhang et al., 1992.. The cleavage rate was determined 48 h after the exposure of the in vitro matured oocytes to spermatozoa during IVF and embryos were
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examined for development on Days 6, 8 and 10 of culture using an inverted microscope at 100 = . 3. Experimental design 3.1. Experiment 1 Effects of treatment of spermatozoa with resazurin ŽR-7017, Sigma. on fertilization of oocytes and subsequent embryo development were investigated using a randomized complete block design. A total of 2767 oocytes was used. Each of the 11 replicates consisted of oocytes from the same collection of abattoir ovaries. The concentration of Percoll gradient-separated motile spermatozoa was adjusted to 50 = 10 6 motile sperm cellsrml and exposed to 17.6 m g resazurinrml in Sperm-TALP ŽParrish et al., 1988. for 0 ŽControl., 15 ŽTRT2., 30 ŽTRT3. and 60 min ŽTRT4., respectively. After treatment of spermatozoa with resazurin for the specific periods of time, 20 m l aliquots of the sperm suspension were added to 500 m l Fert-TALP containing IVM matured oocytes in plastic 4-well petri dishes ŽNunclon w , Nunc. covered with paraffin oil, and incubated in a humidified 5% CO 2 atmosphere at 398C for 17 h. The fertilized ova were then cultured and embryo development evaluated as described above. The concentration of resazurin and duration of treatment with resazurin was scheduled according to the resazurin reduction assay for spermatozoa as described by Glass et al. Ž1991. and Dart et al. Ž1994.. 3.2. Experiment 2 Effects of resazurin redox dye during IVM, IVF and IVC of bovine ova were compared in terms of oocyte cleavage, morula and blastocyst production, using a randomized complete block design. A total of 1671 bovine oocytes was used. Each of the six replicates consisted of oocytes from the same collection of abattoir ovaries. Four Table 1 Development of in vitro fertilized bovine embryos with spermatozoa treated with resazurin for different durations in time prior to fertilization Treatment durationa Žmin.
Oocytes matured
Oocyte cleavage rate at 48 hc Ž%.
Number and percentage Ž%. b of cleaved ova matured cleavage that developed to: Morulae at Blastocysts at Expanded and Day 6 d Day 8 hatching at Day 10
0 15 30 60
637 721 691 718
541Ž84.9. 627Ž87.0. 594Ž86.0. 623Ž86.8.
236Ž43.6. 255Ž40.7. 242Ž40.7. 269Ž43.2.
109Ž20.1. 127Ž20.3. 121Ž20.4. 130Ž20.9.
71Ž13.1. 79Ž12.6. 79Ž13.3. 85Ž13.6.
a The in vitro matured bovine oocytes were fertilized in vitro with spermatozoa treated with 17.6 m g resazurinrml for 0, 15, 30 and 60 min, respectively. b The percentage of each treatment in this table represents 11 replications. c 0 h s the time when the in vitro matured oocytes were added to Fert-TALP containing spermatozoa. d Day 0 s IVF.
Treatment ŽTRT. 1.8 m g resazurinrml in: Oocytes matured IVM TRT1 TRT2 TRT3 TRT4 CTRL 1
q4 y y q y
1
IVF y5 q y q y
2
IVC y y q q y
Oocyte cleavage rate at 48 h))
3
335 339 344 341 312
248Ž74.0. 278Ž82.0. 282Ž82.0. 260Ž76.2. 255Ž81.7.
Number and percentage Ž%.) of cleaved ova that develop to: Morulae at Day 6)))
Blastocysts at Day 8
Expanded and hatching at Day 10
84Ž33.9. 122Ž43.9. 100Ž35.5. 81Ž31.2. 98Ž38.4.
31Ž12.5. a 42Ž15.1. a 18Ž6.4. b 9Ž3.5. b 44Ž17.3. a
17Ž6.9. a 17Ž6.1. a 3Ž1.1. b 1Ž0.4. b 26Ž10.2. a
IVMs in vitro maturation. 2 IVF s in vitro fertilization. 3 IVC s in vitro culture. q4 indicates that the in vitro process was executed in the medium containing 1.8 m g resazurinrml. y5 indicates that the in vitro process was executed in the medium not containing resazurin. )The percentage of each treatment in this table represents six replications. ))0 h s the time when the in vitro matured oocytes were added to Fert-TALP containing spermatozoa. )))Day 0 s IVF. a,b Means within columns without common superscripts differ Ž P - 0.05..
S. Wang et al.r Animal Reproduction Science 51 (1998) 205–213
Table 2 Development of bovine embryos treated with resazurin during in vitro maturation, fertilization and culture
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treatments were used ŽTable 2.: TRT1—oocytes were matured in medium containing 1.8 m g resazurinrml, fertilized and cultured in medium containing no resazurin; TRT2 —oocytes were matured in medium containing no resazurin, followed by IVF in medium containing 1.8 m g resazurinrml and IVC in medium containing no resazurin; TRT3—oocytes were matured and fertilized in medium containing no resazurin, followed by IVC in medium containing 1.8 m g resazurinrml. TRT4—oocytes were matured, fertilized and cultured in media containing 1.8 m g resazurinrml. Oocytes that were matured, fertilized and cultured in medium containing no resazurin, served as the control ŽCTRL.. Because the culture period was 24 h for IVM, 17 h for IVF and the IVC lasted several days, the concentration of resazurin was reduced from 17.6 m grml in Experiment 1 to 1.8 m grml Ž7 m M. in Experiment 2. The reduced concentration of resazurin is still within the usable range to detect reduction color changes ŽFang et al., 1997.. 3.3. Statistical analysis Percentage data were angularly transformed and analyzed by the use of a general linear model ŽGLM. ANOVA. The Fisher’s Least Significant Difference ŽLSD. at 5% significant level Ž P - 0.05. was used to test the differences between treatments. The Number Cruncher Statistical System ŽNCSS. Version 5.01 computer software package ŽHintze, 1988. was used for all statistical analyses. 4. Results In Experiment 1, where bovine oocytes were fertilized with spermatozoa treated with resazurin for 60 min or less prior to IVF, there was no difference Ž P ) 0.05. with respect to oocyte cleavage rate, or morula and blastocyst production as a percentage of cleaved oocytes among treatments ŽTable 1.. In Experiment 2, there was no difference Ž P - 0.05. in cleavage rates among groups when oocytes were exposed to resazurin during IVM, IVF, IVC alone, or during the entire IVM, IVF and IVC culture period ŽTable 2.. However, post-cleavage embryo development was significantly Ž P - 0.05. altered by treatment of oocytesrembryos with resazurin during IVC or IVMrIVFrIVC ŽTRT3, 4, Table 2.. As shown in Table 2, the proportion of IVMrIVF-derived ova that developed into blastocysts and into expanded and hatched blastocysts with TRT3 and TRT4 was less Ž P - 0.05. than that in the control group or in those treated with resazurin only during IVM ŽTRT1., or only during IVF ŽTRT2.. 5. Discussion Neither the fertilizing capacity as determined by oocyte cleavage rates, nor the potential of subsequent embryo development as determined by morula and blastocyst production were altered by treatment of sperm to resazurin ŽTable 1.. The results presented in Table 1 also indicate that treatment of spermatozoa for 60 min with resazurin at the concentration at which color changes could be easily detected is not detrimental to sperm fertilizing capacity and subsequent embryo development. The
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concentration of resazurin and incubation time are two critical factors that determine the accuracy of the resazurin test for predicating fertility of spermatozoa. According to Glass et al. Ž1991. and Dart et al. Ž1994., resazurin added at a final concentration of 25 m grml to a sample of bovine semen results in measurable color changes within 15 min. However, in practice, the false-negative rate is too high if the reduction time is less than 30 min, while false-positive results increase with incubation times longer than 60 min ŽGlass et al., 1991; Dart et al., 1994.. Therefore, incubation for 1 h appears to be the most accurate for sperm assays ŽGlass et al., 1991.. Our results indicate that resazurin has no significant adverse effects on fertilizing capacity and subsequent development of bovine embryos at the recommended concentration and incubation time for the resazurin sperm assay. Results from Experiment 2 show that exposure oocytes to resazurin during IVM ŽTRT1, Table 2. or during IVF ŽTRT2, Table 2. did not significantly retard embryonic development, while preimplantation embryo development is sensitive to resazurin as indicated by reduced numbers of ova developing to the blastocyst stage ŽTRT3, 4, Table 2.. Though both the dosage regimens of resazurin in the IVC medium and duration of treatment time may be related with retarded embryo development, the detrimental effects mainly depended on the period of time for the treatment with resazurin. Bovine oocytes are routinely matured for 24 h, fertilization incubation for 17 h and post-fertilization embryo culture may last for days depending on the research purposes ŽSirard et al., 1988; Bavister, 1995.. These schedules were followed in the present experiments. The duration of treatment of the embryos with resazurin lasted for 10 days during IVC, which is much longer than the duration of treatment during either IVM or IVF. Therefore, the lengthy period of treatment of embryos with resazurin was most likely responsible for the detrimental effects on development in the present study. The negative effects had also been documented in earlier applications of resazurin for assessing fertility in bulls which had limited success due to greater than optimal concentrations of dye and lengthy incubation periods ŽDart et al., 1994.. To use resazurin as a non-invasive assessment of embryo viability, finding a concentration of dye and a suitable reduction time are important factors to be considered. Because cell numbers of preimplantation embryos are a relatively small fraction of cells compared with the numbers of spermatozoa used in a resazurin sperm assay, the incubation time has to be increased to detect any resazurin reduction. Fang et al. Ž1997. observed a detectable reduction product Žresorufin. at a concentration of 0.31 m M, which may be a way to lessen the detrimental effects of resazurin by decreasing the dye concentration in culture medium. Decreasing duration of treatment with resazurin to shorter intervals, for example, 24 h and establishing a minimal, but optimal, concentration of resazurin will be helpful in developing a test for non-invasively assessing embryo viability.
6. Conclusions Resazurin had no adverse effects on in vitro fertilizing capacity of spermatozoa or in vitro development of resulting embryos when used at the recommended dosage and
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duration of treatment for the assay of semen quality. Therefore, resazurin can be used for the assessment of semen quality prior to use in IVF. However, the extended period of treatment of zygotes with resazurin in culture in vitro reduced survival and development of embryos.
Acknowledgements This research was supported by grants from the Utah Department of Agriculture agreement number 90-2720 and USDA-APHIS-VS agreement number 94-9-1-49-0233-C and by the Utah Agricultural Experiment Station, Utah State University, Logan. Approved as journal paper no. 10030. The authors thank E.A. Miller, Hyrum, UT, for the donation of ovaries.
References Bavister, B.D., 1995. Culture of preimplantation embryos: facts and artifacts. Hum. Reprod. Update 1 Ž2., 91–148. Carter, R.A., Ericsson, S.A., Corn, C.D., Weyerts, P.R., Escue, S.G., Mesta, J., 1995. Determination of the fertility potential of equine semen samples using the reducible dyes methylene green and resazurin. Proceedings of the Society for the Study of Theriogenology, p. 316. Comhaire, F.H., Vermeulen, L., 1995. Human semen analysis. Hum. Reprod. Update 1 Ž4., 343–362. Comhaire, F.H., Vermeulen, L., Schoonjans, F., 1987. Reassessment of accuracy of traditional sperm characteristics and adenosine triphosphate ŽATP. in estimating the fertilizing potential of human semen in vivo. Int. J. Androl. 41, 653–662. Cooper, T.A., Wang, S., Liu, Y., Bunch, T.D., Holyoak, G.R., 1995. A new method to evaluate the viability of cryopreserved ram semen using a resazurin reduction assay. Theriogenology 45 Ž1., 314, Abstr. Dart, M.G., Mesta, J., Crenshaw, C., Ericsson, S.A., 1994. Modified resazurin reduction test for determining the fertility potential of bovine spermatozoa. Arch. Androl. 33 Ž2., 71–75. Erb, R.E., Ehler, F.H., 1952. Modified resazurin reduction test for estimating fertilizing capacity of bull semen. J. Dairy Sci. 35, 881–888. Fang, W.H., Myllys, V., Sandholm, M., 1997. Resazurin reduction as a function of respiratory burst in bovine neutrophils. Am. J. Vet. Res. 58, 601–607. Fuse, H., Okumura, M., Kazama, T., Katayama, T., 1993. Comparison of resazurin test results with various sperm parameters. Andrologia 25 Ž3., 153–157. Glass, R.H., Drouin, M.T., Ericsson, S.A., Marcoux, L.J., Ericsson, R.J., Sullivan, H., 1991. The resazurin reduction test provides an assessment of sperm activity. Fertil. Steril. 56, 743–746. Hawk, H.W., Wall, R.J., 1994. Improved yields of bovine blastocysts from in vitro-produced oocytes: I. Selection of oocytes and zygotes. Theriogenology 41 Ž8., 1571–1583. Hinting, A., Comhaire, F.H., Schoonjans, F., 1988. Capacity of objectively assessed sperm motility characteristics in differentiating between semen of fertile and subfertile men. Fertil. Steril. 50, 635–639. Hintze, J.L., 1988. Number Cruncher Statistical System, Version 5.01, 1988. Kaysville, Utah. Mahmoud, A.M., Comhaire, F.H., Vermeulen, L., Andreou, E., 1994. Comparison of the resazurin test, adenosine triphosphate in semen, and various sperm parameters. Hum. Reprod. 9 Ž9., 1688–1693. Mattila-Sandholm, T., Ali-Vehmas, T., Wirtanen, G., Ronner, U., Sandholm, M., 1991. Automated fluorimetry in quality control of pasteurized and ultra-high temperature-treated starch soup. Int. J. Food Sci. Tech. 26, 325–336. Parrish, J.J., Susko-Parrish, J., Winer, M.A., First, N.L., 1988. Capacitation of bovine sperm by heparin. Biol. Reprod. 38, 1171–1180.
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Sirard, M.A., Parrish, J.J., Ware, C.B., Leibfried-Rutledge, M.L., First, N.L., 1988. The culture of bovine oocytes to obtain developmentally competent embryos. Biol. Reprod. 39, 546–552. Wang, S., Liu, Y., Cooper, T., Liu, G., Holyoak, G.R., 1996. Evaluation of the viability of ram spermatozoa by resazurin reduction and spectrophotoscopy. Biol. Reprod. 54, 90, Suppl. Abstr. Wang, S., Liu, Y., Holyoak, G.R., Bunch, T.D., 1997. The influence of M-199 and a modified CR-2 medium on the in vitro production of bovine embryos. Theriogenology 47 Ž1., 304, Abstr. Zalata, A., Hafez, T., Mahmoud, A., Comhaire, F.H., 1995. Relationship between resazurin reduction test, reactive oxygen species generation, and gamma-glutamyltransferase. Hum. Reprod. 10 Ž5., 1136–1140. Zhang, L., Denniston, R.S., Godke, R.A., 1992. A simple method for in vitro maturation, in vitro fertilization and co-culture of bovine oocytes. J. Tissue Culture Methods 14, 107–112.
Effects of resazurin on bovine oocyte fertilization and embryo development in vitro S. Wang a
a,)
, G.R. Holyoak a , G. Liu a,b, T.J. Bunch c , R.C. Evans a , T.D. Bunch a
Department of Animal, Dairy and Veterinary Sciences, Utah State UniÕersity, Logan, UT 84322-9400, USA b Department of Biotechnology, Northeast Agricultural UniÕersity, Harbin 150030, China c UniÕersity of Utah School of Medical, Salt Lake City, UT 84113, USA Accepted 20 February 1998
Abstract Resazurin is a redox dye Ž7-hydroxy-3H-phenoxazin-3-one-10-oxide. used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation ŽIVM., fertilization ŽIVF. and culture ŽIVC.. In Experiment 1, bovine oocytes Ž n s 2767. were fertilized with spermatozoa exposed to resazurin Ž17.6 m grml. for 0, 15, 30, 60 min, respectively. There was no significant Ž P ) 0.05. difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes Ž n s 1671. were treated with resazurin Ž1.8 m grml. during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant Ž P ) 0.05. difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytesrembryos were treated with resazurin during IVC or IVMrIVFrIVC was significantly Ž P - 0.05. less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with
)
Corresponding author: Tel.: q1 435 797 3970; fax: q1 435 797 3494; e-mail: [email protected]
0378-4320r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 0 7 2 - 4
206
S. Wang et al.r Animal Reproduction Science 51 (1998) 205–213
resazurin may be detrimental to embryonic development. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cattle—reproductive technology; Resazurin; Oocyte maturation; In vitro fertilization; Embryo culture
1. Introduction Resazurin is a redox dye Ž7-hydroxy-3H-phenoxazin-3-one-10-oxide. traditionally used for assessing microbiological quality of milk and dairy products ŽMattila-Sandholm et al., 1991.. Because the reduction of resazurin is characterized by color changes, and because the intermediate of the resazurin reduction reaction is a strong fluorogenic redox indicator Žresorufin., minor changes in concentration of resazurin can be detected by photometric or fluorometric methods ŽMattila-Sandholm et al., 1991.. This chemical property of resazurin dye allows for potential uses to quantitatively study the functional status of eukaryotic cells ŽFang et al., 1997.. Resazurin has been used to measure the metabolic activity of spermatozoa and to assess fertilizing capacity ŽErb and Ehler, 1952; Glass et al., 1991; Dart et al., 1994; Carter et al., 1995.. When activated by metabolically viable spermatozoa, resazurin is first reduced to resorufin and then to dihydroresorufin. The reduction reaction is manifested by visual changes in dye color from blue Žresazurin. to pink Žresorufin. and further to white Ždihydroresorufin. and is measurable quantitatively by spectrophotometry ŽWang et al., 1996.. The rate of color change associates to the potential fertilizing capacity of spermatozoa. Usually, the faster the color change from blue to pink and to white, the better the semen quality ŽGlass et al., 1991; Dart et al., 1994.. The resazurin sperm assay mainly depends on the concentration of live sperm ŽMahmoud et al., 1994; Zalata et al., 1995.. As a test for fertility, the assay correlates better than most sperm characteristics commonly used in routine semen analysis ŽComhaire et al., 1987; Fuse et al., 1993; Mahmoud et al., 1994; Zalata et al., 1995.. Resazurin is also a more reliable measurement of metabolic activity for sperm quality than ATP measurement ŽHinting et al., 1988; Comhaire and Vermeulen, 1995.. The simplicity and accuracy of the resazurin assay has been supported by the data from fertility tests in humans ŽGlass et al., 1991. and several domestic animals ŽDart et al., 1994; Comhaire and Vermeulen, 1995; Carter et al., 1995; Cooper et al., 1995.. Due to its sensitivity and reproductivity as an indicator of dehydrogenase activity in the metabolic assessment of cell viability ŽMattila-Sandholm et al., 1991., resazurin has the potential to be used in non-invasive assessments of semen quality and embryo viability. However, there is a lack of information on the effects of resazurin on fertilization and the effects on embryo development. Recent advances in in vitro maturation ŽIVM., in vitro fertilization ŽIVF. and in vitro embryo culture ŽIVC. provide an analytical methodology for assessing fertilizing capacity of spermatozoa and the developmental potential of preimplantation embryos ŽBavister, 1995.. The effects of resazurin can, therefore, be evaluated by the use of in vitro models. The objectives of this study were Ž1. to examine the effects of resazurin treatment on fertilizing capacity of spermatozoa using the technique of in vitro production of bovine
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embryos and Ž2. to investigate the effects of resazurin on the development of bovine oocytesrembryos during oocyte maturation, oocyte fertilization and embryo development in vitro.
2. Materials and methods Ovaries were collected from a local abattoir. Oocytes were aspirated from small antral follicles Ž3–8 mm in diameter. as described by Hawk and Wall Ž1994.. Cumulus oocyte complexes ŽCOCs. with evenly granulated ooplasm and surrounded by several layers Žat least 3 to 4 layers. of compact cumulus cells were selected for use according to the oocyte grading system described by Hawk and Wall Ž1994.. Oocytes were washed three times with HEPES-TALP solution ŽParrish et al., 1988. and once with maturation medium. The maturation medium consisted of M-199 plus 10% Žvolrvol. fetal bovine serum ŽFBS., 25 mM HEPES, 2 mM glutamine, 0.25 mM sodium pyruvate, 0.5 m grml ovine FSH ŽF-4520, Sigma, St. Louis, MO, USA., 5.0 m grml ovine LH ŽL-5269, Sigma. 1.0 m grml estradiol ŽE-2258, Sigma.. In vitro maturation ŽIVM. of oocytes followed the procedure of Sirard et al. Ž1988.. Polystyrene plastic 4-well culture petri dishes ŽNunclon w , Nunc, Naperville, IL, USA. were used for IVM culture. Each well contained 500 m l IVM medium covered with paraffin oil. Approximately 40 to 65 oocytes were transferred to the IVM medium and cultured in a humidified 5% CO 2 atmosphere at 398C for 24 h. Cryopreserved bovine semen was used for in vitro fertilization ŽIVF.. Live sperm were separated by Percoll gradients Ž45% and 90% on the upper and lower layers, respectively. and centrifuged at 500 = g for 30 min. Motile spermatozoa were added to the fertilization medium ŽFert-TALP, Parrish et al., 1988. to provide a final concentration of 2 = 10 6 per ml. Capacitation of spermatozoa occurred in Fert-TALP containing 10 m g heparinrml and 0.6% fatty acid free bovine serum albumin. IVM matured oocytes were added to Fert-TALP containing spermatozoa and cultured in plastic 4-well petri dishes ŽNunclon w , Nunc. under paraffin oil in a humidified 5% CO 2 atmosphere at 398C for 17 h. Each well contained 500 m l Fert-TALP and approximately 40 to 65 oocytes. Cumulus and corona cells were removed from ova by vortexing in HEPES-TALP supplemented with 0.3% bovine serum albumin for 3 min. The presumptive zygotes were then cultured in plastic 4-well petri dishes ŽNunclon w , Nunc. under paraffin oil at 398C in a humidified 5% CO 2 atmosphere. A modified CR2 medium ŽWang et al., 1997. comprising 108.3 mM NaCl, 2.9 mM KCl, 24.9 mM NaHCO 3 , 2.5 mM hemicalcium lactate, 0.5 mM sodium pyruvate, BME amino acids ŽB-6766, Sigma., MEM non-essential amino acids ŽM-7145, Sigma., 0.5 mM glycine, 0.5 mM alanine, 1.0 mM glutamine, 1.0 mM glucose, and antibiotics was used to culture embryos. Each well contained 500 m l CR2 medium with approximately 40 to 60 oocytes. During culture, medium was changed every other day so that it was supplemented with 5% Žvolrvol. fetal bovine serum on Day 1 ŽDay 0 s IVF., 10% on Day 3, 15% on Day 5 and 20% on Day 7 of culture ŽZhang et al., 1992.. The cleavage rate was determined 48 h after the exposure of the in vitro matured oocytes to spermatozoa during IVF and embryos were
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examined for development on Days 6, 8 and 10 of culture using an inverted microscope at 100 = . 3. Experimental design 3.1. Experiment 1 Effects of treatment of spermatozoa with resazurin ŽR-7017, Sigma. on fertilization of oocytes and subsequent embryo development were investigated using a randomized complete block design. A total of 2767 oocytes was used. Each of the 11 replicates consisted of oocytes from the same collection of abattoir ovaries. The concentration of Percoll gradient-separated motile spermatozoa was adjusted to 50 = 10 6 motile sperm cellsrml and exposed to 17.6 m g resazurinrml in Sperm-TALP ŽParrish et al., 1988. for 0 ŽControl., 15 ŽTRT2., 30 ŽTRT3. and 60 min ŽTRT4., respectively. After treatment of spermatozoa with resazurin for the specific periods of time, 20 m l aliquots of the sperm suspension were added to 500 m l Fert-TALP containing IVM matured oocytes in plastic 4-well petri dishes ŽNunclon w , Nunc. covered with paraffin oil, and incubated in a humidified 5% CO 2 atmosphere at 398C for 17 h. The fertilized ova were then cultured and embryo development evaluated as described above. The concentration of resazurin and duration of treatment with resazurin was scheduled according to the resazurin reduction assay for spermatozoa as described by Glass et al. Ž1991. and Dart et al. Ž1994.. 3.2. Experiment 2 Effects of resazurin redox dye during IVM, IVF and IVC of bovine ova were compared in terms of oocyte cleavage, morula and blastocyst production, using a randomized complete block design. A total of 1671 bovine oocytes was used. Each of the six replicates consisted of oocytes from the same collection of abattoir ovaries. Four Table 1 Development of in vitro fertilized bovine embryos with spermatozoa treated with resazurin for different durations in time prior to fertilization Treatment durationa Žmin.
Oocytes matured
Oocyte cleavage rate at 48 hc Ž%.
Number and percentage Ž%. b of cleaved ova matured cleavage that developed to: Morulae at Blastocysts at Expanded and Day 6 d Day 8 hatching at Day 10
0 15 30 60
637 721 691 718
541Ž84.9. 627Ž87.0. 594Ž86.0. 623Ž86.8.
236Ž43.6. 255Ž40.7. 242Ž40.7. 269Ž43.2.
109Ž20.1. 127Ž20.3. 121Ž20.4. 130Ž20.9.
71Ž13.1. 79Ž12.6. 79Ž13.3. 85Ž13.6.
a The in vitro matured bovine oocytes were fertilized in vitro with spermatozoa treated with 17.6 m g resazurinrml for 0, 15, 30 and 60 min, respectively. b The percentage of each treatment in this table represents 11 replications. c 0 h s the time when the in vitro matured oocytes were added to Fert-TALP containing spermatozoa. d Day 0 s IVF.
Treatment ŽTRT. 1.8 m g resazurinrml in: Oocytes matured IVM TRT1 TRT2 TRT3 TRT4 CTRL 1
q4 y y q y
1
IVF y5 q y q y
2
IVC y y q q y
Oocyte cleavage rate at 48 h))
3
335 339 344 341 312
248Ž74.0. 278Ž82.0. 282Ž82.0. 260Ž76.2. 255Ž81.7.
Number and percentage Ž%.) of cleaved ova that develop to: Morulae at Day 6)))
Blastocysts at Day 8
Expanded and hatching at Day 10
84Ž33.9. 122Ž43.9. 100Ž35.5. 81Ž31.2. 98Ž38.4.
31Ž12.5. a 42Ž15.1. a 18Ž6.4. b 9Ž3.5. b 44Ž17.3. a
17Ž6.9. a 17Ž6.1. a 3Ž1.1. b 1Ž0.4. b 26Ž10.2. a
IVMs in vitro maturation. 2 IVF s in vitro fertilization. 3 IVC s in vitro culture. q4 indicates that the in vitro process was executed in the medium containing 1.8 m g resazurinrml. y5 indicates that the in vitro process was executed in the medium not containing resazurin. )The percentage of each treatment in this table represents six replications. ))0 h s the time when the in vitro matured oocytes were added to Fert-TALP containing spermatozoa. )))Day 0 s IVF. a,b Means within columns without common superscripts differ Ž P - 0.05..
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Table 2 Development of bovine embryos treated with resazurin during in vitro maturation, fertilization and culture
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treatments were used ŽTable 2.: TRT1—oocytes were matured in medium containing 1.8 m g resazurinrml, fertilized and cultured in medium containing no resazurin; TRT2 —oocytes were matured in medium containing no resazurin, followed by IVF in medium containing 1.8 m g resazurinrml and IVC in medium containing no resazurin; TRT3—oocytes were matured and fertilized in medium containing no resazurin, followed by IVC in medium containing 1.8 m g resazurinrml. TRT4—oocytes were matured, fertilized and cultured in media containing 1.8 m g resazurinrml. Oocytes that were matured, fertilized and cultured in medium containing no resazurin, served as the control ŽCTRL.. Because the culture period was 24 h for IVM, 17 h for IVF and the IVC lasted several days, the concentration of resazurin was reduced from 17.6 m grml in Experiment 1 to 1.8 m grml Ž7 m M. in Experiment 2. The reduced concentration of resazurin is still within the usable range to detect reduction color changes ŽFang et al., 1997.. 3.3. Statistical analysis Percentage data were angularly transformed and analyzed by the use of a general linear model ŽGLM. ANOVA. The Fisher’s Least Significant Difference ŽLSD. at 5% significant level Ž P - 0.05. was used to test the differences between treatments. The Number Cruncher Statistical System ŽNCSS. Version 5.01 computer software package ŽHintze, 1988. was used for all statistical analyses. 4. Results In Experiment 1, where bovine oocytes were fertilized with spermatozoa treated with resazurin for 60 min or less prior to IVF, there was no difference Ž P ) 0.05. with respect to oocyte cleavage rate, or morula and blastocyst production as a percentage of cleaved oocytes among treatments ŽTable 1.. In Experiment 2, there was no difference Ž P - 0.05. in cleavage rates among groups when oocytes were exposed to resazurin during IVM, IVF, IVC alone, or during the entire IVM, IVF and IVC culture period ŽTable 2.. However, post-cleavage embryo development was significantly Ž P - 0.05. altered by treatment of oocytesrembryos with resazurin during IVC or IVMrIVFrIVC ŽTRT3, 4, Table 2.. As shown in Table 2, the proportion of IVMrIVF-derived ova that developed into blastocysts and into expanded and hatched blastocysts with TRT3 and TRT4 was less Ž P - 0.05. than that in the control group or in those treated with resazurin only during IVM ŽTRT1., or only during IVF ŽTRT2.. 5. Discussion Neither the fertilizing capacity as determined by oocyte cleavage rates, nor the potential of subsequent embryo development as determined by morula and blastocyst production were altered by treatment of sperm to resazurin ŽTable 1.. The results presented in Table 1 also indicate that treatment of spermatozoa for 60 min with resazurin at the concentration at which color changes could be easily detected is not detrimental to sperm fertilizing capacity and subsequent embryo development. The
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concentration of resazurin and incubation time are two critical factors that determine the accuracy of the resazurin test for predicating fertility of spermatozoa. According to Glass et al. Ž1991. and Dart et al. Ž1994., resazurin added at a final concentration of 25 m grml to a sample of bovine semen results in measurable color changes within 15 min. However, in practice, the false-negative rate is too high if the reduction time is less than 30 min, while false-positive results increase with incubation times longer than 60 min ŽGlass et al., 1991; Dart et al., 1994.. Therefore, incubation for 1 h appears to be the most accurate for sperm assays ŽGlass et al., 1991.. Our results indicate that resazurin has no significant adverse effects on fertilizing capacity and subsequent development of bovine embryos at the recommended concentration and incubation time for the resazurin sperm assay. Results from Experiment 2 show that exposure oocytes to resazurin during IVM ŽTRT1, Table 2. or during IVF ŽTRT2, Table 2. did not significantly retard embryonic development, while preimplantation embryo development is sensitive to resazurin as indicated by reduced numbers of ova developing to the blastocyst stage ŽTRT3, 4, Table 2.. Though both the dosage regimens of resazurin in the IVC medium and duration of treatment time may be related with retarded embryo development, the detrimental effects mainly depended on the period of time for the treatment with resazurin. Bovine oocytes are routinely matured for 24 h, fertilization incubation for 17 h and post-fertilization embryo culture may last for days depending on the research purposes ŽSirard et al., 1988; Bavister, 1995.. These schedules were followed in the present experiments. The duration of treatment of the embryos with resazurin lasted for 10 days during IVC, which is much longer than the duration of treatment during either IVM or IVF. Therefore, the lengthy period of treatment of embryos with resazurin was most likely responsible for the detrimental effects on development in the present study. The negative effects had also been documented in earlier applications of resazurin for assessing fertility in bulls which had limited success due to greater than optimal concentrations of dye and lengthy incubation periods ŽDart et al., 1994.. To use resazurin as a non-invasive assessment of embryo viability, finding a concentration of dye and a suitable reduction time are important factors to be considered. Because cell numbers of preimplantation embryos are a relatively small fraction of cells compared with the numbers of spermatozoa used in a resazurin sperm assay, the incubation time has to be increased to detect any resazurin reduction. Fang et al. Ž1997. observed a detectable reduction product Žresorufin. at a concentration of 0.31 m M, which may be a way to lessen the detrimental effects of resazurin by decreasing the dye concentration in culture medium. Decreasing duration of treatment with resazurin to shorter intervals, for example, 24 h and establishing a minimal, but optimal, concentration of resazurin will be helpful in developing a test for non-invasively assessing embryo viability.
6. Conclusions Resazurin had no adverse effects on in vitro fertilizing capacity of spermatozoa or in vitro development of resulting embryos when used at the recommended dosage and
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duration of treatment for the assay of semen quality. Therefore, resazurin can be used for the assessment of semen quality prior to use in IVF. However, the extended period of treatment of zygotes with resazurin in culture in vitro reduced survival and development of embryos.
Acknowledgements This research was supported by grants from the Utah Department of Agriculture agreement number 90-2720 and USDA-APHIS-VS agreement number 94-9-1-49-0233-C and by the Utah Agricultural Experiment Station, Utah State University, Logan. Approved as journal paper no. 10030. The authors thank E.A. Miller, Hyrum, UT, for the donation of ovaries.
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