Effects of Glutamine, Glycine and Taurine on the Development of In Vitro Fertilized Bovine Zygotes in a Chemically Defined Medium

Transcription

1 Effects of Glutamine, Glycine and Taurine on the Development of In Vitro Fertilized Bovine Zygotes in a Chemically Defined Medium Yoshiyuki TAKAHASHI and Hiroshi KANAGAWA Laboratory of Theriogenology, Department of Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo , Japan (Received 12 June 1997/Accepted 1 December 1997) ABSTRACT. The effects of glutamine, glycine and taurine on the development of bovine zygotes derived from IVM/IVF were determined using a protein-free chemically defined medium. After the cumulus cells were removed at 18 hr post-insemination, the presumptive zygotes were cultured for 4 or 6 days (about 104 or 154 hr) under a gas atmosphere of 5% O 2 : 5% CO 2 : 90% N 2. A modified synthetic oviduct fluid medium supplemented with 20 amino acids (1 mm glutamine, essential amino acids for basal medium Eagle and nonessential amino acids for minimum essential medium), insulin, and PVA was used as a basic medium (msofai). Omitting 1 mm glutamine from msofai did not affect the embryonic development after 4 and 6 days of culture. After 4 days of culture, no significant effects of glycine and taurine on the development of zygotes to the morula stage were observed. However, supplementation with glycine or taurine significantly (P<0.05) affected, with no interaction, the embryonic development to blastocysts after 6 days of culture. Addition of 5 mm glycine and 2 or 10 mm taurine significantly (P<0.05) increased the percentage of blastocysts. The mean cell number in the blastocysts was affected by the glycine level, and was increased by the addition of 10 mm glycine (P<0.001). These results demonstrate that glycine and taurine in a chemically defined medium containing a group of essential and non-essential amino acids improve the development of bovine zygotes to the blastocyst stage under 5% O 2. KEY WORDS: bovine embryo, culture, glutamine, glycine, taurine. J. Vet. Med. Sci. 60(4): , 1998 Adequate information about the requirements of bovine embryos is essential for the study of embryo development and for the in vitro production of bovine embryos using in vitro maturation (IVM) and in vitro fertilization (IVF) techniques. Recent attention has been focused on the effects of specific substances such as energy substrates, amino acids and growth factors. It has been demonstrated that exogenous supplementation with amino acids is important for the development of mammalian embryos [1, 2]. Glutamine, glycine and taurine have been found to be particularly important for the in vitro development of hamster embryos [1, 2]. Glutamine is preferentially metabolized by bovine embryos during the 2- to 4-cell stages [25]. Glycine and taurine are the most abundant amino acids in the bovine oviductal and uterine fluids [6, 21, 27]. Therefore, these three amino acids seem to play important roles in the development of bovine embryos. The effects of glutamine and glycine on bovine embryonic development have been investigated by several groups, however, the supplementation of media with glutamine [10, 12, 20, 23, 33] or glycine [12, 15, 21, 28] has given inconsistent results. One of the reasons is that the culture conditions used in these studies were variable: complex vs. simple media, with vs. without other amino acids, presence vs. absence of bovine serum albumin (BSA), and so on. For studying the specific requirements for early embryonic development, a protein-free chemically defined medium is necessary to eliminate unknown stimulatory or inhibitory substances usually included in the serum and BSA [2]. More than 20 amino acids are present in the bovine oviductal and uterine fluids [6, 21, 27]. The study using a defined medium supplemented with a group of amino acids similar to those in the reproductive tract fluids seems to be representative of the in vivo conditions. The oxygen tensions in the oviduct and uterus were reported to be much less than half of the atmospheric oxygen tension [7]. It is well known that the incubation under 20% O 2 is harmful for embryonic development, and that an oxygen concentration of 5 to 10% in the gas atmosphere is optimal for the development of bovine embryos when they are cultured in vitro without feeder cells [31, 32]. Taurine, serving as an oxidant or an oxygen radical scavenger, has been found to counteract the negative effect of 20% O 2, but the addition of taurine was not beneficial under 5% O 2 on the development of bovine 4- to 8-cell embryos [14]. The addition of hypotaurine, which is the precursor of taurine, has recently been reported to improve bovine embryonic development even under 5% O 2 [8]. The effect of taurine is still obscure, and needs to be determined. In the present study, the effects of the above-mentioned major amino acids, glutamine, glycine and taurine, on the development of bovine zygotes derived from IVM/IVF were examined using a protein-free chemically defined medium containing a group of amino acids (essential amino acids for basal medium Eagle and nonessential amino acids for minimum essential medium) in a gas atmosphere of 5% O 2 : 5% CO 2 : 90% N 2. MATERIALS AND METHODS Source of zygotes: Bovine zygotes were obtained after IVM and IVF as described previously [31]. Briefly, bovine cumulus-oocyte complexes (COCs) were aspirated from small antral follicles on the ovaries obtained from a

2 434 Y. TAKAHASHI AND H. KANAGAWA slaughterhouse. To accomplish IVM, they were cultured for 22 hr in 50-µl drops (10 to 13 COCs per drop) of HEPES-buffered TCM199 (Gibco Laboratories, Grand Island, NY, U.S.A.) supplemented with 10% heat-treated fetal calf serum (Gibco), 0.02 units/ml FSH (Sigma Co., St. Louis, MO, U.S.A.), 1 µg/ml estradiol-17β (Sigma), 0.2 mm pyruvate (Sigma) and 50 µg/ml gentamicin (Sigma). IVF was performed using frozen semen from two Holstein bulls donated by the Hokkaido Livestock Improvement Association (Hokkaido, Japan). Semen from the same bull was used for each experiment, and after thawing, motile sperm were separated using a Percoll gradient (45 and 90%). The COCs were co-incubated for 18 hr with sperm ( cells/ml) in 100-µl drops (10 to 13 COCs per drop) of modified Brackett and Oliphant s (BO) medium containing 3 mg/ml fatty acid-free BSA (Sigma) and 2.5 mm theophylline (Sigma) [30]. Cultures for IVM and IVF were maintained at 39 C in humidified air with 5% CO 2 using a CO 2 incubator (Napco 510, Napco Scientific Co., Tualtin, OR, U.S.A.). In the preliminary experiments, the frequency of in vitro fertilization had resulted in more than 85% sperm penetration of oocytes and around 80% of normal fertilization by using semen from both bulls. In vitro culture of zygotes: After co-incubation with sperm, the presumptive zygotes were stripped of cumulus cells by vortex agitation in HEPES-buffered Tyrode s medium [3]. The cumulus-free zygotes were washed four times with each culture medium. They were then incubated for another 4 days (about 104 hr) or 6 days (about 154 hr) at 39 C in 40-µl drops (25 to 40 zygotes per drop) covered with paraffin oil under a gas atmosphere of 5% O 2 with 5% CO 2 and 90% N 2 using an oxygen-adjustable N 2 -O 2 -CO 2 incubator (BPN 110, Tabai Espec Co., Osaka, Japan). A modified synthetic oviduct fluid medium supplemented with 20 amino acids, 10 µg/ml insulin (from bovine pancreas, Sigma) and 1 mg/ml polyvinyl alcohol (PVA, Sigma), designated as msofai [31], was used as a basal medium. One mm glutamine (L-glutamine, Sigma) and premixture solutions of 12 essential amino acids for basal medium Eagle (EAA-BME, Flow Laboratories, Irvine, Scotland) and 7 nonessential amino acids for minimum essential medium (NEAA-MEM, Flow) were added to provide 20 amino acids. The premixture solution of nonessential amino acids contained 0.1 mm glycine. The embryonic development to the morula and blastocyst stages was examined under a stereomicroscope either Day 5 (about 122 hr) or Day 7 (about 172 hr) after insemination. Experimental studies: To determine the influence of glutamine on embryonic development, presumptive zygotes were cultured for 4 days (Experiment 1a) or 6 days (Experiment 1b) in msofai containing 0 or 1 mm glutamine. In Experiment 2a, presumptive zygotes were cultured for 4 days in msofai and msofai with glycine (5.1 or 10.1 mm) or taurine (2 or 10 mm), and development to the morula stage was studied. Experiment 2b was a 3 3 factorial design comparing 3 levels of glycine (0.1, 5.1, and 10.1 mm) and taurine (0, 2, and 10 mm) on embryo development to the blastocyst stage after 6 days of culture. All embryos beyond the 8-cell stage (Experiments 1a and 2a) and all blastocysts (Experiments 1b and 2b) were assigned for cell counting by an air-dry procedure [29]. The embryos possessing 16 cells or more were judged as morulae as described previously [29, 31]. Statistical analysis: In Experiments 1a and 1b, the differences in the data (percentages of cleaved zygotes, morulae and blastocysts, and cell numbers of morulae and blastocysts) between the two groups were analyzed by Student s t-test. In Experiment 2a, comparisons were made by Fisher s protected least significant difference (PLSD) test after one-way analysis of variance (ANOVA). The data in Experiment 2b were analyzed by two-way ANOVA followed by Fisher s PLSD as post-hoc test. RESULTS Experiment 1: As shown in Table 1, there was no significant difference in the embryonic development with or without glutamine after 4 and 6 days of culture (P>0.1). The cell counts, which were performed on all morulae and blastocysts to provide another treatment effect, also demonstrated the lack of effect of glutamine. Experiment 2: When zygotes were cultured for 4 days (Experiment 2a), the supplementation with glycine and taurine had no detectable effect on the cleavage rate, percentage of embryos developing into morulae, and the mean cell numbers (P>0.1) as shown in Table 2. When presumptive zygotes were cultured for 6 days (Experiment 2b), two-way ANOVA revealed that there was no significant interaction between the effects of glycine and taurine (P>0.1), and that both the glycine and taurine levels affected the embryonic development to blastocysts (P<0.05 and P<0.01, respectively). As shown in Table 3, glycine at 5.1 mm (P<0.05) and taurine at 2 or 10 mm (P<0.01) increased the percentage of blastocysts. The total cell number per blastocyst was affected only by the glycine level, and was increased by supplementation with 10.1 mm glycine (P<0.001). DISCUSSION The present study revealed that glutamine at 1 mm had neither a beneficial nor a deleterious effect on the development of bovine embryos. It also demonstrated that the addition of 5 mm glycine or 2 to 10 mm taurine improved the in vitro development of bovine zygotes to the blastocyst stage after 6 days of culture in a protein-free chemically defined medium, although no significant effects on the development to the morula stage were detected. The beneficial effect of glutamine on embryonic development has been reported in hamsters [17], mice [4], pigs [22], and cattle [20]. In the present study, however, no beneficial effect of glutamine in combination with essential amino acids for basal medium Eagle (EAA-BME) and nonessential amino acids for minimum essential medium

3 GLYCINE AND TAURINE ON BOVINE EMBRYOS 435 Table 1. Effect of supplementation of msofai with glutamine (Gln) on the in vitro development of bovine zygotes to morulae and blastocysts (Experiments 1a and 1b) Exp. Conc. No. of % of % of Total cell No. per No. Gln zygotes cleaved morulae blastocysts morula blastocyst (mm) (replicates) zygotes at Day 5 a) at Day 7 b) (N) (N) ± ± ± a (5) (44) ± ± ± 19.0 (5) (49) ± ± ± b (5) (29) ± ± ± 81.4 (5) (26) Values are means ± SD. a) and b) % based on the number of cleaved zygotes about 122 hr and 172 hr after insemination, respectively. Table 2. Effects of glycine (Gly) and taurine (Tau) on in vitro development of bovine zygotes to the morula stage (Experiment 2a) Concentration No. of % of % of Total cell No. (mm) of zygotes cleaved morulae at per Gly Tau (replicates) zygotes Day 5 a) morula (N) (5) 83.4 ± ± ± 22.1 (57) (5) 81.7 ± ± ± 23.2 (70) (5) 79.2 ± ± ± 20.2 (54) (5) 81.6 ± ± ± 17.1 (66) (5) 82.8 ± ± ± 17.0 (57) Values are means ± SD. a) % based on the number of cleaved oocytes at 122 hr after insemination. (NEAA-MEM) was observed even in the absence of glucose. This result supports the recent finding that the addition of 1 mm glutamine singly or in combination with EAA-MEM and/or NEAA-MEM, does not improve bovine embryo development [10, 12, 33]. Further experiments are needed to determine the optimal concentrations of glutamine for in vitro culture and its role in the development of bovine embryos, since it has been demonstrated that bovine embryos utilize glutamine as an energy source throughout their preimplantation development [25], and that the response of mouse embryos to glutamine depended on the concentration of NaCl in the culture medium [11]. Moore and Bondioli [21] first reported the beneficial effect of glycine at 10 mm concentration on the development of bovine embryos in a modified Brinster s ovum culture medium containing BSA and NEAA-MEM with and without oviductal cells. The addition of 10 mm glycine to CR2 medium containing EDTA and BSA [28] or to synthetic oviduct fluid medium (SOF) with EAA-MEM [12] also improved bovine embryonic development. In contrast, supplementation with 10 mm glycine has been reported to have no beneficial effect on the development of bovine embryos to blastocysts when the embryos were cultured in SOF in combination with EAA-MEM and NEAA-MEM [12]. Moreover, the addition of 10 mm glycine to KSOM containing EDTA, 0.5x concentration of EAA-MEM and 1x NEAA-MEM reduced the blastocyst formation rate of bovine 4- to 8-cell embryos [15]. Lee and Fukui [12] tested the dose effect of glycine supplemented to SOF with PVA and EAA-MEM. However, there has been no report on the optimum concentration of glycine in a defined medium supplemented with both EAA and NEAA. The concentrations of glycine in the fluids of cow oviduct and uterus were reported to be 1 to 2.4 mm [21, 27] and 1 to 5 mm [6], respectively. Therefore, the present results suggest that the optimum concentration of glycine may be 5 mm or less to enhance the development of bovine zygotes to blastocysts in combination with EAA-BME and NEAA- MEM. In the addition of 10 mm glycine, an increase in the total cell number per blastocyst was observed irrespective of improvement in the rate of blastocyst formation. It is likely that 10 mm of glycine might be too high to support the development of embryos at the oviductal stage, but glycine could enhance the development of uterine stage embryos under the present culture conditions. Supplementation of media with taurine has been demonstrated to enhance in vitro development of mouse [5, 26], hamster [17], rabbit [13], pig [24], and cattle embryos [16]. Most of these studies have performed embryo culture in a gas atmosphere of 5% CO 2 in air (approximately 20%

4 436 Y. TAKAHASHI AND H. KANAGAWA Table 3. Effects of glycine (Gly) and taurine (Tau) on in vitro development of bovine zygotes to the blastocyst stage (Experiment 2b) Concentration No. of % of % of Total cell No. (mm) of zygotes cleaved blastocysts per blastocyst Gly Tau (replicates) zygotes at Day 7 a) (N) (5) 84.7 ± ± 6.6 b) ± 60.9 b,c) (47) (5) 81.5 ± ± 5.0 c) ± 60.6 b,c) (55) (5) 84.5 ± ± 9.0 c) ± 60.4 c) (71) (5) 82.6 ± ± 4.6 c,d) ± 58.9 c) (52) (5) 82.1 ± ± 7.8 c) ± 53.6 b,c) (57) (5) 81.3 ± ± 4.9 c) ± 45.8 b,c) (55) (5) 85.2 ± ± 4.0 b,d) ± 70.5 d) (46) (5) 82.2 ± ± 5.5 c,d) ± 51.5 b,c) (47) (5) 83.7 ± ± 6.2 c,d) ± 55.3 b,d) (61) Overall means (15) 83.5 ± ± 9.1 e) ± 60.3 g) (173) (15) 82.0 ± ± 5.7 f) ± 52.8 g) (164) (15) 83.7 ± ± 5.3 e) ± 59.6 h) (154) Overall means (15) 84.2 ± ± 6.9 g) ± 65.4 (145) (15) 81.9 ± ± 6.5 h) ± 55.3 (159) (15) 83.2 ± ± 7.0 h) ± 55.5 (187) Values are means ± SD. a) % based on the number of cleaved zygotes at 172 hr postinsemination. b,c,d) Values within columns for individual treatments with different superscripts differ (P<0.05). e,f) and g, h) Main effects means within columns with different superscripts differ (P<0.05 and P<0.01, respectively). O 2 ). Liu and Foote [14] showed that the inclusion of 7 or 14 mm taurine improved the development of 4- to 8-cell bovine embryos to the blastocyst stage after 6 days of culture in 20% O 2, but that taurine was not beneficial in 5% O 2. Most recently, Fujitani et al. [8] reported that the addition of 10 mm hypotaurine to TCM199 supplemented with 1% calf serum increased the cell number in the blastocyst, although hypotaurine did not improve the percentage of bovine 4- to 6-cell embryos that developed to the blastocyst stage. In the present experiment, the addition of 2 or 10 mm taurine enhanced the development of zygotes to blastocysts even in a gas atmosphere with 5% O 2. The susceptibility of embryos to suboptimal culture conditions is higher in the earlier developmental stages than in the advanced stages [2]. The pharmacological effects of taurine are observed in the stress system [9]. Thus, the discrepancy might be attributable to the difference in the developmental stages of embryos at the initiation of culture (4- to 8-cell embryos vs. zygotes). It is also possible that the efficacy of taurine may vary according to the composition of the basal medium employed, since taurine can serve not only as an oxidant or free radical scavenger, but also serve as an osmolyte or chelating agent [1, 2, 9]. Alternatively, taurine might modulate the beneficial effects of insulin that enhances bovine embryonic development by stimulating amino acids transport [16, 18]. The basic medium (msofai) used in the present study contained insulin, and taurine has been reported to bind to insulin receptors and appears to mediate some of the physiological actions of insulin [19]. The beneficial effects of glycine and taurine on embryonic development appeared after 6 days of culture, although significant effects were not detectable after 4 days of culture. This is probably due to their latent effects rather than their possible effects on the embryonic development only during the advanced stage because the in vitro culture of bovine embryos after the cell-block stage (8- to 16-cell) is relatively easier than that from earlier stages [2]. There was no significant interaction between the effect of glycine and that of taurine suggesting that they might affect embryonic development in different ways. The actual mechanisms by which glycine and taurine exert their beneficial roles in the development of bovine embryos remain to be elucidated. In conclusion, the present experiments demonstrate that 1 mm glutamine does not affect embryonic development, and that addition of 5 mm glycine or 2 to 10 mm taurine improves the in vitro development of bovine zygotes to the blastocyst stage in a protein-free chemically defined medium supplemented with EAA-BME and NEAA-MEM under a gas atmosphere of 5% O 2 : 5% CO 2 : 90% N 2. ACKNOWLEDGMENTS. This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and by grants from the Ito Foundation and the Morinaga Hoshi-Kai to

5 GLYCINE AND TAURINE ON BOVINE EMBRYOS 437 YT. The authors thank the staff of the Ebetsu Meat Inspection Office for the collection of abattoir materials, and Hokkaido Livestock Improvement Association for the donation of frozen bull semen. REFERENCES 1. Barnett, D. H. and Bavister, B. D What is the relationship between the metabolism of preimplantation embryos and their developmental competence? Mol. Reprod. Dev. 43: Bavister, B. D Culture of preimplantation embryos: factors and artifacts. Hum. Reprod. Update 1: Bavister, B. D., Leibfried, M. L. and Lieberman, G Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol. Reprod. 28: Chatot, C. L., Tasca, R. J. and Ziomek, C. A Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium. J. Reprod. Fertil. 89: Dumoulin, J. C. M., Evers, J. L. H., Bras, M., Pieters, M. H. E. C. and Geraedts, J. P. M Positive effect of taurine on preimplantation development of mouse embryos in vitro. J. Reprod. Fertil. 94: Fahning, M. L., Schultz, R. H. and Graham, E. F The free amino acid content of uterine fluids and blood serum in the cow. J. Reprod. Fertil. 13: Fischer, B. and Bavister, B. D Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J. Reprod. Fertil. 99: Fujitani, F., Kasai, K., Ohtani, S., Nishimura, K., Yamada, M. and Utsumi, K Effect of oxygen concentration and free radicals on in vitro development of in vitro-produced bovine embryos. J. Anim. Sci. 75: Huxtable, R. J Physiological actions of taurine. Physiol. Rev. 72: Keskintepe, L., Burnley, C. A. and Brackett, B. G Production of viable bovine blastocysts in defined in vitro conditions. Biol. Reprod. 52: Lawitts, J. A. and Biggers, J. D Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media. Mol. Reprod. Dev. 31: Lee, E-S. and Fukui, Y Synergistic effect of alanine and glycine on bovine embryos cultured in a chemically defined medium and amino acid uptake by in vitro-produced bovine morulae and blastocysts. Biol. Reprod. 55: Li, J., Foote, R. H. and Simkin, M Development of rabbit zygotes cultured in protein-free medium with catalase, taurine, or superoxide dismutase. Biol. Reprod. 48: Liu, Z. and Foote, R.H Development of bovine embryos in KSOM with added superoxide dismutase and taurine and with five and twenty percent O 2. Biol. Reprod. 53: Liu, Z. and Foote, R. H Effects of amino acids and α- amanitin on bovine embryo development in a simple protein-free medium. Mol. Reprod. Dev. 46: Liu, Z., Foote, R. H. and Yang, X Development of early bovine embryos in co-culture with KSOM and taurine, superoxide dismutase or insulin. Theriogenology 44: McKiernan, S. H., Clayton, M. K. and Bavister, B. D Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro. Mol. Reprod. Dev. 42: Matsui, M., Takahashi, Y., Hishinuma, M. and Kanagawa, H Stimulatory effects of insulin on the development of bovine embryos fertilized in vitro. J. Vet. Med. Sci. 57: Maturo, J. and Kulakowski E. C Turine binding to the purified insulin receptor. Biochem. Pharmacol. 37: Moore, K. and Bondioli, K. R Altering metabolic substrates of culture media enhances bovine IVM/IVF embryo development beyond the eight-cell block. Biol. Reprod. 42 (Suppl. 1): 55 (abstr. 43). 21. Moore, K. and Bondioli, K. R Glycine and alanine supplementation of culture medium enhances development of in vitro matured and fertilized cattle embryos. Biol. Reprod. 48: Petters, R. M., Johnson, B. H., Reed, M. L. and Archibong, A. E Glucose, glutamine and inorganic phosphate in early development of the pig embryo in vitro. J. Reprod. Fertil. 89: Pinyopummintr, T. and Bavister, B. D Energy substrate requirements for in vitro development of early cleavage-stage bovine embryos. Mol. Reprod. Dev. 44: Reed, M. L., Illera, M. J. and Petters, R. M In vitro culture of pig embryos. Theriogenology 37: Rieger, D., Loskutoff, N. M. and Betteridge, K. J Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and co-cultured in vitro. J. Reprod. Fertil. 95: Spindle, A Beneficial effects of taurine on mouse zygotes developing in protein-free culture medium. Theriogenology 44: Stanke, D. F., Sikes, J. D., DeYoung, D. W. and Tumbleson, M. E Proteins and amino acids in bovine oviductal fluid. J. Reprod. Fertil. 38: Suh, T. K., White, K. L., Bunch, T. D., Spendlove, R. and Wilkinson, R Effect of glycine, alanine and calf plasma in serum free culture medium on bovine embryonic development in vitro. Theriogenology 43: 328 (abstr.). 29. Takahashi, Y. and First, N. L In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 37: Takahashi, Y. and First, N. L In vitro fertilization of bovine oocytes in the presence of theophylline. Anim. Reprod. Sci. 34: Takahashi, Y., Hishinuma, M., Matsui, M., Tanaka, H. and Kanagawa, H Development of in vitro matured/fertilized bovine embryos in a chemically defined medium: influence of oxygen concentration in the gas atmosphere. J. Vet. Med. Sci. 58: Thompson, J. G. E., Simpson, A. C., Pugh, P. A., Donnelly, P. E. and Tervit, H. R Effect of oxygen concentration on in-vitro development of preimplantation sheep and cattle embryos. J. Reprod. Fertil. 89: Yoshioka, K., Takahashi, Y., Hishinuma, M. and Kanagawa, H In vitro culture of bovine one-cell embryos derived from in vitro fertilization using a semi-chemically defined medium. J. Vet. Med. Sci. 55: