In Vitro Developmental Competence of In Vitro-Matured Bovine
Oocytes
Fertilized and Cultured in Completely Defined Media
Levent Keskintepe and Benjamin G. Brackett2Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602-7389
ABSTRACT
The objective was to establish an in vitro system in which bovine oocytes can be matured, fertilized, and cultured up to the blastocyst stage without support of serum, BSA, or somatic cells. Media consisted of modified tissue culture medium 199 (mTCM 199) with ovine LH (oLH) for maturation (IVM), exper-imental alterations of modified defined medium (mDM) for sperm selection and insemination (IVF), and citrate + synthetic oviductal fluid + nonessential amino acids (c-SOF+NEA) for zy-gote/embryo culture (IVC). Effects of heparin, BSA, polyvinyl alcohol (PVA), penicillamine (P), Hepes, and sodium bicarbon-ate (NaHCO3) were studied. Results included proportions of
oo-cytes that cleaved by 48 h and that reached morulae by 120 h, blastocysts by 168 h, and expanded blastocysts by 216 h postin-semination (pi). Best results were obtained when the IVF me-dium included 0.5 mg P + 1.0 mg PVA per milliliter with no more than 10 mM Hepes, and when 3.0 mg PVA/ml and 10 mM Hepes were present for IVC. Different concentrations of Na-HCO3, up to 50 mM from 25 mM, during IVF did not alter
results. Embryos produced in defined conditions yielding the best results remained viable after vitrification as evidenced by continued development in vitro for 96 h postthawing. Bovine oocytes matured in defined medium supplemented with LH were fertilized and cultured up to the blastocyst stage in chem-ically defined conditions that afforded results comparable to those reported earlier after inclusion of serum, BSA, and/or so-matic cells.
INTRODUCTION
The development of defined bovine embryo culture sys-tems holds promise for ongoing efforts in reproductive physiology and for utility in the embryo transfer industry. Although a variety of embryo culture systems are employed for in vitro embryo production, systems that include ovi-duct epithelial cells [1-3], granulosa cells [4-6], or tropho-blastic vesicles [2, 7] are lacking adequate definition to as-sure quality control and repeatability. To eliminate exces-sive variability, and to lead to better understanding of preimplantational development, simplified culture systems have been employed [8-14]. Recent reports on defined in vitro conditions have assisted the understanding of molec-ular mechanisms capable of promoting or impeding early embryonic development [15-17]. Amino acids [8, 10, 13,
18-20], energy substrates [21, 22], citrate [13, 23], and vi-tamins [10, 22, 24] can profoundly influence embryonic de-velopment in vitro.
Serum and BSA are among the most common compo-nents of media for mammalian embryo culture. Serum, which contains hormones, growth factors, vitamins,
chela-Accepted March 21, 1996. Received December 28, 1995.
'This work was supported by funds from Transgenic Products, Inc., Atlanta, GA.
2Correspondence. FAX: (706) 542-3015; e-mail: brackett.b@calc.vet.uga.edu
tors for heavy metal ions, peptides, proteins, and an array of defined and nondefined molecules [25], is generally in-cluded as the fixed nitrogen source for the preimplantation embryo [17]. However, serum was found to have a biphasic influence on development of bovine embryos, being dele-terious to the first cleavage division but stimulatory for blastocyst development [11, 26]. In early mouse embryo experiments, BSA ameliorated water quality when embryo-toxicity was a problem [27]. BSA may be contaminated with several extraneous peptides, energy substrates, and growth factors [28, 29]. Different batches of commercially available BSA might inhibit or stimulate embryonic devel-opment [23, 26]. BSA has been commonly replaced by syn-thetic macromolecules, polyvinyl alcohol (PVA) or poly-vinylpyrrolidone (PVP), when chemically defined condi-tions are desired [13, 30, 31].
Among other components investigated during in vitro maturation, fertilization, and culture (IVMFC) are sodium bicarbonate (NaHCO3) [32, 33]; insulin, transferrin, and
se-lenium [34]; growth factors [35, 36]; gas atmosphere [37]; glucose [38]; penicillamine (P) [39]; antioxidants [40]; and water quality [41]. However, completely defined conditions have been unavailable for definitive testing; either serum or BSA has been included for one or more of the steps composing the IVMFC procedure. All incompletely defined biological additives including heparin treatment for sperm capacitation should be eliminated for a more accurate as-sessment of influences of experimental treatments on IVMFC outcome.
The objectives of this study were to define conditions for sperm treatment, in vitro fertilization (IVF), and in vitro culture (IVC) without compromising the quantity and qual-ity of blastocyst production from immature oocytes via the entire IVMFC system. Influences of heparin, BSA, PVA, P, NaHCO3, and Hepes during IVF and IVC were studied, and
products of IVMFC were challenged by vitrification to as-sess competency for continuing in vitro development.
MATERIALS AND METHODS
Oocyte Recovery and In Vitro Maturation (IVM)
Oocyte collection and IVM were carried out as reported earlier [13]. Briefly, bovine ovaries were obtained within 15-30 min after slaughter. Follicles 2-5 mm in diameter were aspirated with a 10-cc syringe fitted with an 18-gauge needle. Follicular aspirates were collected into a 50-ml plastic flask (Falcon 3014; Becton Dickinson Labware, Franklin Lakes, NJ) and then held at 30-33°C during trans-port to the laboratory (-2.5 h). Oocyte selection [42] and all other manipulations were carried out at 39C. Oocytes with 3-4 layers of cumulus cells and having homogenous cytoplasm were washed through PBS with 0.4 mg/ml poly-vinyl alcohol (P-8136; Sigma Chemical Co., St. Louis, MO) and transferred into 100-il drops of mTCM 199 (M-3769, Sigma) with additions for IVM during a 24-h 333
TABLE 1. Experimental design and treatment groups.a
Experi- Sperm swim-up in IVC in
ment mDMb with IVF in mDM with c-SOF + NEA with 1 Hepesc + 6.0 mg Hepes + 6.0 mg PVA Hepes + 6.0 mg
PVA or BSA or BSA (Control)/ml PVA/ml (control)/ml + P (0, 0.5, 2.0, 4.0
mg/ml)
2 Hepes + 6.0 mg Hepes + 0.5 mg P/ml Hepes + 6.0 mg PVA/ml + PVA (0, 1.0, 3.0, PVA/ml
6.0 mg/ml)
3 Hepes + 1.0 mg Hepes + 0.5 mg P/ml Hepes + PVA (0,
PVA/ml + 1.0 mg PVA/ml 1.0, 3.0, 6.0
mg/ml)
4 Hepes + 1.0 mg 0.5 mg P/ml + 1.0 mg Hepes + 3.0 mg
PVA/ml PVA/ml + Hepes (0, PVA/ml
10, 25 mM)
5 1.0 mg PVA/ml 0.5 mg P/ml + 1.0 mg 3.0 mg PVA/ml +
PVA/ml Hepes (0, 10,
25 mM)
6 0.5 mg P/ml + 1.0 0.5 mg P/ml + 1.0 mg 3.0 mg PVA/ml +
mg PVA/ml PVA/ml + NaHCO3 10 mM Hepes
(25, 37, 50 mM)
a Oocytes were matured in vitro as described in Materials and Methods. Salient features are in bold type; alterations adopted appear in italics.
b Defined medium [43] was modified by deletion of BSA and addition of 1.0 mg/ml caffeine prior to experimental additions indicated.
c Hepes concentration was 25 mM except where absent or otherwise indicated.
interval. Additions to mTCM 199 for IVM were 50 }.g/ml sodium pyruvate (P-2256, Sigma), 2.6 mg/ml sodium bi-carbonate (S-5761, Sigma), 50 pVg gentamicin sulfate (G-1264, Sigma), and 100 ,ag/ml oLH (ovine LH-26, BIO, lot no. AFP-5551B; NHPP, NIDDK, NICHD, USDA). In-cubations for IVM were carried out under paraffin oil (S894-00; J.T. Baker Inc., Phillipsburg, NJ) and a moist 5% C02:5% 02:90% N2 atmosphere.
Sperm Preparation and IVF
Sperm washing, swim-up, and insemination (IVF) were performed in defined medium (DM) [43] with use of PVA (P-8136, Mr 30 000-70 000; Sigma) as a macromolecule. Amounts varied depending on experiment (Table 1). Me-dium in the control group (experiment 1) contained 0.6% BSA in place of PVA. (See Table I for concentrations of P [P-4875, Sigma], PVA, and Hepes used in each experi-ment.) Caffeine (1.0 mg/ml, C-0750; Sigma) was added for fertilization throughout. In each experiment, two straws containing frozen semen from one fertile bull (108 sperm/ 0.5-ml straw; Atlantic Breeders Cooperative, Lancaster, PA) were thawed in a 37°C water bath for 30 sec; 0.1 ml of thawed semen was layered under 1.5 ml of medium in each of several small tissue culture tubes (12 75 mm; Fisher Scientific, Pittsburgh, PA) and held at a 45° angle for 45 min at 39°C. Then the top 0.8 ml from each tube was removed, pooled in a sterile 15-ml centrifuge tube, and centrifuged (320 x g) for 10 min. In experiments 1-5, each resulting sperm pellet was resuspended to 200 Rl with hep-arin (200 i.g/ml, sodium salt, H-7005; Sigma) in a micro-centrifuge tube (5 x 10 mm). In experiment 6, pellets were similarly resuspended but without heparin.
Mature oocytes were randomly assigned to groups of 15 in 100-1I drops to which sperm cells were added 15 min later. Sperm were counted in a hemacytometer and checked for motility (i.e., at least 80% progressively motile); then oocytes were inseminated (by addition of 10-12 p1 sperm suspension) for a final concentration of 2.0 x 106 motile sperm/ml. Sperm and oocytes were coincubated for IVF
under paraffin oil and moist 5% CO2:5% 02:90% N2 for
24 h.
IVC
Culture of presumptive zygotes and early 2-cell-stage embryos was initiated, as described earlier [13, 39], in 50 1l of BSA-free synthetic oviduct fluid (SOF) with additions of PVA (refer to Table 1 for concentrations), 0.5 mM citrate ("sodium citrate-trisodium salt", S-4641; Sigma), and MEM nonessential amino acids (M-7145, Sigma), abbre-viated as c-SOF+NEA. Embryonic development was mon-itored, and 50% of the culture volume was replaced with fresh medium at 24-h intervals. Proportions of cleaved oo-cytes were recorded 48 h after insemination. Oooo-cytes that did not progress to the next cleavage stage were removed from drops containing developing embryos at the time of each subsequent medium change. Proportions of insemi-nated oocytes reaching morulae were recorded at 120 h; blastocysts, at 168 h; and expanded blastocysts, at 216 h postinsemination (pi).
Embryo Cryopreservation
Freezing of late morula or early blastocyst stages was carried out by a vitrification procedure, originally described for mouse embryos [44] and modified for bovine embryos [45]. All treatments before freezing were carried out at room temperature (22°C). Bovine embryos (late morula and early blastocyst stages) removed from culture at 144 h pi were exposed for 3 min to 20% ethylene glycol (E-9129, Sigma) in embryo-holding medium, a modified phosphate buffer solution supplemented with 0.33 mM sodium pyru-vate, 5.6 mM glucose, and 0.4% BSA (cat. no. 3320-60; Intergen Company, Purchase, NY). Then embryos were ex-posed for 30-45 sec to a vitrification solution consisting of 40% (v:v) ethylene glycol (Sigma), 18% (w:v) Ficoll (F-2878, Sigma), and 10.26% (w:v) sucrose (S-5 79115; Fisher Scientific Company, Fair Lawn, NJ). Groups of 5 embryos were then loaded into 0.5-ml French straws and
FIG. 1. Effects of varying P concentrations (0, 0.5, 2.0, 4.0 mg/ml) in mDM for IVF of in vitro-matured bovine oocytes. Results are reported as per-centages of oocytes selected for IVMFC that reached 2- to 4-cell (C),
morula (M), blastocyst (B), and expanded blastocyst (EB) stages within the indicated intervals pi. Dissimilar superscripts within stages denote signif-icant differences (at indicated P values).
vitrified by directly plunging into liquid nitrogen (LN2).
After storage intervals in LN2for 24 h to 96 h, single straws
were thawed in water at 25C. Contents were immediately diluted in a solution of 0.25 mM sucrose, and embryos were transferred into 50-p1 drops of Menezo B2 (INRA
Menezo B2 Medium; bioMerieux, Marey-l Etoile 69280, France) supplemented with 20% fetal bovine serum (FBS; S1150; Atlanta Biologicals, Norcross, GA). Frozen-thawed embryos were cultured for up to 120 h under oil to assess
continued viability. Blastocyst Staining
Blastocysts were stained with Hoechst 33342 stain [46]. Groups of 5 blastocysts were removed from culture at 216 h pi and transferred to a slide in 2-3 1l of medium. Then,
15 lI of Hoechst stain prepared with Na citrate (2.3%) and ethyl alcohol was added. The slide was incubated at 37°C for 5 min; then extra stain was discarded, and Permount (15 1l) was added along with a coverslip. Total cells in each blastocyst were counted under a fluorescence micro-scope (320).
Experimental Studies
Salient features of six successive experiments appear in bold type in Table 1; alterations adopted as a result of prior experimental findings appear in italics.
Defined medium was supplemented with P (0, 0.5, 2.0, or 4.0 mg/ml) during IVF for experiment 1. Endpoint eval-uation was development as described earlier. The effects of various PVA concentrations (0, 1.0, 3.0, 6.0 mg/ml) were evaluated during IVF (experiment 2) and IVC (experiment 3). Bovine oocytes and sperm were incubated in P (0.5 mg/ml)-containing mDM with different concentrations of PVA for 24 h; then oocytes were transferred into c-SOF+NEA containing 6.0 mg PVA/ml until 216 h pi (ex-periment 2). In ex(ex-periment 3, bovine oocytes were incu-bated with sperm in mDM with 0.5 mg P + 1.0 mg PVA per milliliter for 24 h; then oocytes were transferred into c-SOF+NEA with varying PVA concentrations for IVC (to 216 h pi). Various concentrations of Hepes, in mDM (ex-periment 4) and in c-SOF+NEA (ex(ex-periment 5), were stud-ied for influences on IVMFC outcome after incorporation of superior conditions found with respect to P and PVA.
In experiment 6, best conditions found in the previous experiments were applied without heparin treatment for sperm. Different concentrations of NaHCO3 (25, 37, and
FIG. 2. Effect of varying concentration of PVA during in vitro insemination of bovine oocytes. The BSA (6.0 mg/ml)-containing group was included as a standard, and the means were compared by pairwise Bonferroni t-test.
50 mM) were incorporated in the mDM. Some late morulae and early blastocysts (144 h pi), products of defined con-ditions, were frozen by vitrification. For assessment of the ability of these IVMFC embryos to survive this rigorous treatment after thawing, they were subjected to IVC in Me-nezo B2 [45] and attention was paid to survival and sub-sequent developmental progress. Survival data were deter-mined by evidence of development in culture within 72 h.
Statistical Analyses
Each IVMFC experiment was repeated three times, and data were analyzed through use of the SigmaStat (Jandel Scientific, San Rafael, CA) software package. One-way re-peated measures ANOVA was applied to assess statistical differences after data were transformed by arcsin square root method. The Bonferroni t-test was used to determine differences among the groups. Experimental endpoints were proportions of initially selected immature oocytes reaching indicated stages (n/n), recorded also as percentages. Dif-ferences of p < 0.05 were taken as significant.
RESULTS
Experiment 1: Effect of P during IVF
Additions of P to mDM for IVF were assessed for influ-ences on fertilization and early development (Fig. 1). Ad-dition of 0.5 mg/ml P resulted in development of more morulae by 120 h, blastocysts by 168 h pi (p < 0.05), and expanded blastocysts by 216 h pi (p < 0.001) than other concentrations tested.
Although there was no difference in cleavage after use of mDM with 0.5 or 2.0 mg/ml concentrations of P for IVE by 48 h pi the 0 and 4.0 mg/ml treatments yielded lower proportions of cleaved oocytes. The control (6 mg/ml BSA in mDM) supported the highest proportions of cleavage, i.e., 76%, but subsequent development was comparable to that obtained after inclusion of 0.5 mg/ml P in the mDM for IVE
Experiment 2: Effect of PVA Concentration during IVF As shown in Figure 2, the proportions of oocytes that reached 2- to 4-cell stages were not statistically different among the BSA control, 0, 1.0, and 6.0 mg/ml PVA groups; these groups had a higher percentage fertilization rate than 3.0 mg/ml PVA (p < 0.005). Significant increases in pro-portions of oocytes that developed to morula (p < 0.0001), blastocyst (p = 0.0024), and expanded blastocyst (p =
TABLE 3. Effect of Hepes (0, 10, 25 mM) in c-SOF + NEA for IVC on bovine embryonic development.
Number of oocytes/embryos (%) Insem-Hepes inated C M B EB (mM) 0 h 48 h 120 h 168 h 216 h 0 95 76 (80.0), 50 (52.6)c 38 (40.0) 30 (31.6)m 10 97 83 (85.6)b 56 (5 7.7)d 45 (46.4)f 37 (38.1)h 25 95 80 (8 4.2)b 52 (5 4.7)d 40 (42.1)' 33 (34.7)g * Dissimilar superscripts within columns denote differences (p < 0.0001). FIG. 3. Influences of different concentrations of PVA on bovine embryonic
development during in vitro culture from 24 to 216 h pi.
0.0016) stages were obtained in culture after IVF in mDM containing 1.0 mg/ml PVA.
Experiment 3: Effect of PVA Concentration in c-SOF+NEA for IVC
PVA was included (0, 1.0, 3.0, 6.0 mg/ml) in c-SOF+NEA during culture from 24 to 216 h pi (Fig. 3). The proportions of oocytes that developed to 2- to 4-cell, morula, blastocyst, and expanded blastocyst (p < 0.0001) stages were superior when 3.0 mg/ml PVA was present.
Experiment 4: Effect of Hepes Concentration in mDM during IVF
As shown in Table 2, the presence of 10 mM Hepes during IVF had no effect on development to 2- to 4-cell, morula, blastocyst, and expanded blastocyst stages as com-pared with mDM without Hepes. However, the presence of 25 mM Hepes had a detrimental effect on zygote cleavage, morula, blastocyst, and expanded blastocyst development (p < 0.0001).
Experiment 5: Effect of Hepes Concentration in c-SOF+NEA during IVC
As shown in Table 3, 10 mM Hepes in c-SOF+NEA yielded better cleavage and development rates than those observed in the absence of Hepes (p < 0.0001).
Experiment 6: Effect of Varying Concentrations of NaHCO3 in mDM for IVF
Results of experiments to examine effects of three con-centrations of NaHCO3 in mDM during the 24-h
insemi-nation interval are summarized in Table 4. Measured pH and osmolalities at the onset of insemination for each NaHCO3 concentration were 25 mM, pH 7.8, 301 mOsm
per kg; 37 mM, pH 7.8, 327 mOsm per kg; and 50 mM,
TABLE 2. Influences of varying Hepes concentrations (0, 10, 25 mM) in mDM for IVF on bovine embryonic development.
Number of oocytes/embryos (%)* Insem-Hepes inated C M B EB (mM) 0 h 48 h 120 h 168 h 216 h 0 108 95 (87.9)a 67 (62.0)c 48 (44.4)' 40 (37.0)g 10 108 92 (85.2)" 63 (58.3)c 42 (38.8)' 37 (34.3)g 25 110 64 (5 8.2)b 45 (40.9) a 30 (27.3)f 25 (22.7)h
* Dissimilar superscripts within columns denote differences (p < 0.0001).
pH 8.0, 344 mOsm per kg. Results were not affected by altered NaHCO3 concentrations when compared with the
control (37 mM) (p > 0.05). Further, study of 20 represen-tative expanded blastocysts revealed no differences in cell numbers among the groups.
In Vitro Survival of Control Embryos from Experiment 6 after Vitrification
A total of 96 late morulae and early blastocysts (144 h pi) from conditions of experiment 6 (with 37 mM NaHCO3
during IVF) were challenged by vitrification. After thawing, 67.0% of the embryos had survived by 72 h, and 82.0% of these reached the expanded blastocyst stage by 96 h culture (Fig. 4).
DISCUSSION
Results from the present study clearly demonstrate that bovine oocytes can be matured, fertilized, and cultured up to the expanded blastocyst stage without losing their de-velopmental competence in completely defined conditions. BSA could be effectively replaced with PVA when P was present in the fertilization medium (experiment 1). Al-though BSA has been known to effect the sperm acrosome reaction [47], the precise mechanism remains unclear. Dur-ing the acrosome reaction, BSA might act as a metal ion chelator [27, 28, 48]. It has been suggested that D-penicil-lamine, L-histidine, and L-cysteine are able to capacitate hamster spermatozoa in the absence of exogenous proteins [48]; D-penicillamine was found to be the most competent divalent cation chelator for bovine spermatozoa [39].
In a number of studies, P was found to enable an in-crease in the percentage of spermatozoa undergoing the ac-rosome reaction in the presence of epinephrine [49-51]. However, these experiments were carried out in undefined conditions, and the efficacy of being able to induce acro-some reactions was attributed to epinephrine or hypotau-rine, without regard for P. Including 0.5 mg/ml P + PVA has supported proportions of morula, blastocyst, and ex-panded blastocyst development in vitro similar to the those supported by undefined conditions (experiment 1). Our
ear-TABLE 4. Absence of effects of three concentrations of bicarbonate in mDM during IVF on bovine embryonic development (p > 0.05).
Bicarbon-aent-ra No.of No. of embryos No. of nuclei
tion oocytes developing from oocy counted at
during cul- M B EB 216 h
IVF (mM) tured 120 h 168 h 216 h (mean + SEM) 25 93 69 (74.2) 62 (66.7) 57 (61.3) 91.0 + 5.23 37 93 71 (76.3) 64 (68.8) 59 (63.4) 90.6 + 4.16 50 93 70 (75.3) 64 (68.8) 57 (61.3) 91.5 + 6.36
IUJ '0 - a 0 80-60. 40. 20. I100 to .80 > ; Y .60 m .40 E 0a .20 ' 0 O 0 0 24 48 96 120 144
Hours of embryo culture after thawing
-- Percent survival; --- Development to EB
FIG 4. Survival, and development of surviving embryos, after vitrification of 96 embryos at 144 h pi.
lier experiments [13] demonstrated that amino acids (es-sential and/or nones(es-sential) were not supportive of bovine oocyte development after IVF when added during the 24 h in vitro insemination interval.
Spontaneous acrosome reactions were observed when PVA was replaced by BSA in studies in the mouse [52] and hamster [53]. Although PVA has been successfully ployed in oocyte maturation [14] and culture of bovine em-bryos [11, 13, 15], fertilization rates were reduced com-pared to those observed in the presence of BSA [54]. When bovine oocytes were inseminated in a medium that was lacking protein and PVA, no penetration of oocytes fol-lowed [33]. This emphasizes that fertilization requires sup-port of a macromolecular component for insemination. Our results (experiments 1-5) demonstrated that high propor-tions of development could be obtained by employing DM [43] with added caffeine, and could be further modified by replacing BSA by PVA and with the addition of P for IVE It has been suggested that penetration of cumulus-cell-en-closed oocytes starts 5 h after onset of insemination [55] when BSA is used for IVF; however, when PVA was used, penetration could start as early as 1 h after onset of insem-ination [33].
Other laboratories have reported decreased cleavage and blastocyst development rates when PVA was present in the fertilization medium [55]. Similarly, Eckert and Niemann [14] demonstrated that pronuclear formation was retarded when proteins were eliminated from the insemination me-dium; however, protein supplementation was unnecessary for maturation and culture. Contrary to these results, the present study demonstrates high proportions of cleavage and development to the expanded blastocyst stage when PVA concentrations were adjusted for support of IVF (ex-periment 2) and IVC (ex(ex-periment 3).
It has been established that mammalian embryos express stage-specific receptors for specific growth factors and their ligands during development [56-58]. Experimental evi-dence also implies that the factor or factors produced by embryos not only help their own development but also help others in the same culture medium [17]. Therefore, groups of embryos have been cultured in smaller volumes to fa-cilitate this beneficial activity in vitro [17]. During the first 72 h of culture, embryos metabolize amino acids and pro-duce ammonium ions as a result. Generation of high amounts of ammonium ions is extremely toxic to mam-malian embryos [17]. The culture medium must be changed at intervals to protect embryos from this toxicity. It has
been suggested that amino acids for in vitro culture have a role in buffering intracellular pH [59]. However, Hepes and/or NaHCO3are usually added to culture media to assure better
pH regulation [13, 15, 33].
One of the obstacles in gamete manipulation in vitro is loss of CO2 from the NaHCO3-buffered medium, with a
resultant rise in pH [60]. Progress in the development of organic buffers such as Hepes [61] has led to their suc-cessful use in mammalian in vitro systems [62]. Lee and Storey [60] observed no fertilization when they buffered TCM 199 medium with only 25 mM Hepes. However, they observed 70% fertilization when 25 mM Hepes was added to a 10 mM HCO3--containing medium. The results pre-sented here demonstrate that the presence of Hepes in mDM during IVF was not essential; in contrast, it compro-mised bovine embryonic development even though mDM used for fertilization contained both NaHCO3 and Hepes.
Interestingly, 3.0 mg/ml Hepes was an apparently indispen-sable component for c-SOF+NEA to maintain the appro-priate pH (7.4) during in vitro culture.
Bicarbonate was suggested to be a critical component for medium buffering rather than important for a role in acrosome reaction [63]. It was also found that bicarbonate was one of the essential components for hyperactivation of mouse [63] and hamster [64] spermatozoa. Neil and Olds-Clarke [631 found in constant pH conditions that bicarbon-ate might have a different role, for example, in maintaining sperm permeability rather than pH buffering. Tajik et al. [33] obtained high proportions of sperm penetration and female-male pronuclear formation rates with 46 mM NaHCO3 and 1 mg PVA per milliliter. Our results,
evalu-ated in defined conditions, revealed no statistical differ-ences in development after IVF in various NaHCO3
con-centrations (25, 37, 50 mM). Reasons for differences be-tween laboratories may be our inclusion of P for sperm treatment and in vitro insemination and our not using hep-arin for sperm capacitation. Another reason might relate to the salt content of defined medium [43].
This is the first report of completely defined conditions for sperm treatment, IVF, and IVC demonstrated to be ef-ficacious for bovine embryo production. Embryos produced in this system morphologically resemble in vivo-produced embryos under the light microscope; numbers of nuclei were comparable with those reported for in vivo-produced embryos at the same stage, although in vitro development was probably slower [65]. Shamsuddin et al. [65] reported that the accuracy of counts of cell numbers after Hoechst 33342 staining was poor compared to that provided by Giemsa staining. This might account for lower cell numbers than were actually developed in the present work. The sur-vival in vitro that was observed after vitrification of in vitro-produced embryos was similar to that reported by Mahmoudzadeh et al. [45]. Embryo production in defined conditions should open avenues for identifying the factor(s) capable of affecting bovine zygote/embryo development. ACKNOWLEDGMENTS
Authors gratefully acknowledge Atlantic Breeders Cooperative, Lan-caster, PA, for bull semen; National Hormone and Pituitary Program, NIDDK, NICHD, and USDA for oLH; and Shapiro Packing Co., Augusta, GA, for oocytes.
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