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Biology of Reproduction 62, 248-252 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Culture of In Vitro-Produced Bovine Embryos with Vitamin E Improves Development In Vitro and After Transfer to Recipients1

S.E. Olsona, and G.E. Seidel Jr2,a

a Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado 80523


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Detrimental effects of oxygen-derived free radicals on embryos during culture have been demonstrated in several species. Vitamin E occurs naturally in cell membranes and protects cells from oxidative stress. Under some conditions, vitamin C acts synergistically to enhance the antioxidant effects of vitamin E, a benefit that may be further enhanced by EDTA. The present experiments concerned culture of bovine embryos derived from in vitro-matured, fertilized oocytes with vitamin E, vitamin C, and EDTA in a chemically defined culture medium + 0.2% BSA at 5% O2, 5% CO2, and 90% N2. In the first experiment, more zygotes developed to expanded blastocysts (17%, n = 224, P < 0.05) when culture medium contained 100 µM vitamin E than in control medium (11%, n = 234). Development to early, expanded, and hatched blastocysts was lower with vitamins E and C combined than with vitamin E alone (15%, 9%, and 2% vs. 24%, 17%, and 5%, respectively; P < 0.05), as was the mean number of cells per blastocyst (56 vs. 84, P < 0.05). Addition of EDTA (3 µM) failed to improve development over that in culture with vitamin E + vitamin C. In experiment 2, in vitro-produced embryos cultured 5.5 days in medium with or without 100 µM vitamin E were transferred nonsurgically to recipient cows and heifers and then collected nonsurgically 7 days later. Embryos cultured with vitamin E (n = 37) were approximately 63% larger in surface area than controls (1.16 mm2 vs. 0.71 mm2 surface area; n = 27, P < 0.04).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative insults occur to cells in vivo and in vitro from exposure to free radicals generated by exogenous agents (e.g., radiation, chemicals, hyperoxia) and endogenous processes such as normal cellular metabolism. Under extreme oxidative conditions, or if the antioxidant protective mechanisms of cells are compromised, cellular injury and death may occur [1,2]. Early mammalian embryos are susceptible to damage from reactive oxygen species (reviewed in [3]), and they increase the production of oxygen free radicals when cultured in vitro [4]. When free radicals were generated in vitro by addition of 2,2'-azobis(2-amindinopropane) dihydrochloride to culture medium, development of bovine embryos to blastocysts was decreased in a dose-dependent manner [5]. Even sublethal damage may adversely affect embryonic development, resulting in retarded development, perturbation of normal metabolic pathways, and compromised viability.

Vitamin E ({alpha}-tocopherol and derivatives), the predominant lipid-soluble antioxidant in animal cells, protects cells from oxygen radicals in vivo [1,6] and in vitro, and is believed to be the primary free radical scavenger in mammalian cell membranes [7]. Vitamin C (ascorbic acid) is an important water-soluble antioxidant that reduces sulfhydryls and scavenges free radicals. Vitamin C also acts synergistically with vitamin E under some conditions by regenerating tocopherol from tocopheroxyl radicals, the products of tocopherol and oxygen-free radical interaction [8]. However, vitamin C may become a prooxidant agent when free transition metals are present [9]. EDTA, which chelates transition metals, enhances in vitro embryonic development of mouse [10,11] and cattle embryos [12], most likely due to antioxidant effects. EDTA could theoretically enhance the synergy between vitamins E and C by binding metal ions, thus preventing metal-catalyzed reactions that generate reactive oxygen species, in particular highly damaging hydroxyl radicals.

Supplementation of culture media with vitamin E increased survival rates of explanted rat conceptuses in vitro [13] and, as evidenced by vital dye exclusion, increased viability of mouse embryos exposed to heat shock [14]; however, there was no increased development to blastocysts in the latter study.

The objectives of this study were to determine whether vitamin E, with or without vitamin C or with vitamin C and EDTA, improved embryonic development in vitro of bovine embryos produced in vitro as determined by developmental morphology and the number of cells per blastocyst. The second experiment tested whether culture of embryos in vitro with vitamin E improved subsequent growth in vivo after transfer to recipients. Because vitamin E is a naturally occurring molecule that has been widely used for prophylactic protection of other mammalian cells from oxidative injury in vitro, we expected it to be an effective and nontoxic antioxidant for bovine embryo culture.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Oocytes

Bovine ovaries were obtained from slaughtered feedlot heifers and transported at ambient temperature (~16–23°C) approximately 50 km to the laboratory. Ovaries were trimmed of extraneous tissue, rinsed once for 20 sec in 70% ethanol, and then rinsed several times with sterile physiological saline (0.15 M NaCl). Oocytes were aspirated from all visible 2- to 8-mm surface follicles with sterile, washed syringes and 18-gauge hypodermic needles. Selection of oocytes was based on the presence of homogeneous ooplasm and at least several layers of compact cumulus cells.

Maturation and Fertilization of Oocytes

All media were prepared with deionized water, sterile filtered (0.22 µm), and equilibrated in 5% CO2 at 39°C prior to use. Oocytes were matured in Hepes-buffered Tissue Culture Medium (TCM-199, M-2520; Sigma Chemical Co., St. Louis, MO) containing 25 mM NaHCO3, 25 µg/ml gentamicin sulfate, 10% estrous cow serum, 1 µg/ml estradiol-17ß (Sigma; E-8875), 1 µg/ml LH (USDA LH-B-5; Beltsville, MD), and 50 ng/ml FSH (NIH-ovine FSH-17; Bethesda, MD) for 24 h at 39°C in 5% CO2, high-humidity air in 200-µl drops under sterile, washed, paraffin oil. Frozen semen from two bulls was thawed in a 37°C water bath for 20 sec, mixed, and used for in vitro insemination; semen from the same bulls was used throughout the study. Spermatozoa were separated from seminal plasma and extender by a 15-min centrifugation in a 6-ml plastic tube at 600 x g through 1.5 ml Sp-TALP [15] + 5 U/ml heparin (Sp-TALP/hep) + 20% w:v fraction V BSA. The 0.5-ml soft pellet then was resuspended to 5 ml of Sp-TALP/hep, counted with a hemacytometer, and centrifuged 5 min at 600 x g; the 0.5-ml soft pellet was then resuspended to 107 sperm/ml Sp-TALP/hep followed by 2.5 h of incubation at 39°C 5% CO2 in air. For fertilization, 50 µl of sperm suspension was added to oocytes in 200-µl drops of Fert-TALP [15] + 5 mM caffeine under paraffin oil, resulting in 2 x 106 sperm/ml.

Embryo Culture

Experiment 1 After coincubation with sperm for 18 h, ova were vortexed 30 sec in 0.5-ml microcentrifuge tubes containing ~0.25 ml of a chemically defined medium without EDTA (CDM) [16] to remove cumulus cells. Ova were then washed 3 times in 50-µl drops of CDM, and equal numbers (15–25 per replicate) were randomly selected and transferred to Nunc 4-well multidishes (no. 176740; Nunc A/S, Roskilde, Denmark) for culture in CDM + 0.2% fatty acid-free BSA (FAF-BSA; Sigma, A-6003) + 0.05% ethanol (control), or in CDM + 0.2% FAF-BSA supplemented with either 100 µM vitamin E; 100 µM vitamin E + 100 µM vitamin C; or 100 µM vitamin E, 100 µM vitamin C, and 3 µM EDTA. Culture was in 500 µl of medium without paraffin oil in a 5% O2, 5% CO2, 90% N2 humidified atmosphere at 39°C for 7 days. An average of 71% of inseminated oocytes cleaved; uncleaved ova were discarded after 24 h of culture and were not included in this study.

Because vitamin E ([±]-{alpha}-tocopherol; Sigma T-3251) is hydrophobic, it was first dissolved in 95% ethanol as a 2000-strength stock solution, stored in the dark at 4°C, and then (18–20 h prior to embryo culture) diluted in CDM to a final concentration of 100 µM vitamin E and 0.05% ethanol. The small amount of highly purified serum albumin was added to help keep the vitamin E in aqueous solution and facilitate delivery to cell plasma membranes with minimal compromise of the defined nature of the medium. Preliminary experiments indicated that 0.05% ethanol had no effect on bovine embryo development in vitro. Vitamin concentrations were based on approximate physiological levels; normal human plasma ascorbate values are 30–150 µM [17]. The concentration of vitamin E used was ~4–5 times normal human adult plasma levels [18] and in the range of concentrations found effective for in vitro protection of rat hepatocytes (25 µM {alpha}-tocopheryl succinate) [19], rat conceptuses (94 µg/ml or 218 µM {alpha}-tocopherol) [13], and mouse embryos (25, 250 µM {alpha}-tocopheryl succinate) [14]. Because vitamin E is poorly soluble in aqueous solution, and likely mostly bound to BSA in this system, we assumed the actual concentration accessible to the cell to be lower than the amount added. The concentration of EDTA was based on dose-response data from previous experiments with bovine embryos [16].

Experiment 2 Zygotes were cultured as in experiment 1, except that there were only 2 treatments—0 µM (control) and 100 µM vitamin E were used—and culture was terminated after 5.5 days.

Embryo Evaluation

Experiment 1 Embryos were evaluated for morphological stage of development 7 days after being transferred from Fert-TALP to embryo culture media. At that time, blastocysts were stained with Hoechst 33342 to count nuclei with an inverted microscope and epifluorescence using the method of Pursel et al. [20]. Briefly, embryos were exposed to 0.01% trypan blue in water for 30 sec at 39°C; they were then incubated for 12–15 min at 39°C in Hoechst 33342 (10 µg/ml) dissolved in 2.3% sodium citrate and 95% ethanol. Embryos were then mounted on microscope slides, covered with a small drop of Permount (Fisher Scientific, Pittsburgh, PA) slide mounting compound, and gently covered with a coverslip. Slides were either stored in the dark or observed immediately.

Experiment 2 Embryos were evaluated after 5.5 days in culture, and compact morulae or early blastocysts were evaluated by one person and assigned a morphological quality grade [21] ranging from 1 for embryos of excellent morphological quality and showing no evidence of developmental retardation, to 5 for completely degenerate embryos.

Embryo Transfer

Experiment 2 To assay continued viability after in vitro culture, embryos were transferred to recipients for 7 days. Embryos were evaluated 7 days after oocyte collection, and all embryos that were compacted morulae or early blastocysts were transferred. On the basis of data from experiment 1, it was expected that the number of suitable embryos would be similar in each group, approximately 8–10 embryos (starting with 25 oocytes) per treatment per replicate. Embryos from each treatment were transferred nonsurgically with 0.25 ml French straws, using standard techniques for bovine embryo transfer [21], into the uterine horn ipsilateral to the corpus luteum, determined at the time of transfer by palpation per rectum. The number of embryos transferred to each recipient ranged from 1 to 12.

Recipients for Embryo Transfer

Nonlactating crossbred beef cows and heifers were observed twice daily for behavioral estrus. Those selected for this study were seen in standing estrus 12–36 h after oocytes were placed into maturation medium, with preference given to those first observed standing 24 h after the beginning of maturation. For treatments within replicates, available recipients were matched as closely as possible for age and synchrony of estrus.

Embryo Collection

Embryos were collected nonsurgically 7 days after transfer by repeatedly filling and draining the uterus through a Foley catheter with a total volume of approximately 2 L modified Dulbecco's PBS + 0.1% BSA [21]. Embryos were isolated and measured using a calibrated eyepiece micrometer on a stereomicroscope at x15 or x30 magnification.

Statistical Analyses

Experiment 1 was replicated 14 times. Morphological stage of development and number of cells per blastocyst were compared by one-way ANOVA (General Linear Models; SAS Inc., Cary, NC) using least-significant difference procedures to test preplanned comparisons of treatments (1 vs. 2, 2 vs. 3, and 3 vs. 4). Experiment 2 was replicated 16 times. For each replicate, all compacted morulae and early blastocysts from each treatment (range = 1–12 per recipient) were transferred to a single recipient, except in one case in which two recipients were used for each treatment. In one replicate, no embryo was recovered from either recipient. Data from that replicate were included in the calculations of responses determined prior to transfer (e.g., fertilization and development to compacted morulae).

Surface area of viable embryos was calculated using the formulas for a sphere: (surface area = 4{pi}r2) or an ellipsoid (prolate spheroid) according to Mattson et al. [22]. For experiment 2, data on cleavage to two or more cells, development of cleaved ova to compacted morulae, percentage of transferred embryos recovered, percentage of recovered embryos that were viable (development had progressed to at least the expanded blastocyst stage since transfer and embryos had not visibly degenerated), and surface area of viable embryos were analyzed using a one-tail t-test (SAS) to compare effects of culture with and without vitamin E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Supplementation of CDM with 100 µM vitamin E resulted in significantly more expanded blastocysts than in the controls (Table 1). When 100 µM vitamin C was also added, development to early, expanded, and hatched blastocysts, as well as numbers of cells per blastocyst, were lower (P < 0.05) than with vitamin E alone. Development in medium supplemented with EDTA in addition to vitamins E and C was not different (P > 0.1) from that with vitamin E and C alone.


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  		TABLE 1. Effect of vitamin E (E), vitamin C (C), and EDTA on bovine embryo development

Experiment 2

Embryos cultured in vitro with vitamin E had larger mean surface areas (P < 0.04) at collection from recipients than embryos cultured in control medium (Table 2). Approximately 35% of embryos from the control group had surface areas greater than 0.5 mm2, whereas 68% of the vitamin E-cultured embryos were larger than 0.5 mm2 (Fig. 1). There was no difference between control and treated groups in percentages of embryos that cleaved or of cleaved embryos that developed to compacted morulae during culture in vitro (33% and 34%, respectively, P = 0.64). Mean embryo scores, percentage of transferred embryos that were recovered, and percentage of recovered embryos that were viable were also not different between treatments; the total number of embryos transferred per treatment was similar (Table 2). There was no effect of recipient age (heifers or cows) or number of embryos transferred (1–12 per recipient) on size of embryos recovered or efficiency of embryo recovery (P > 0.20; data not presented). Numbers of embryos transferred or recovered from each recipient were not correlated (P > 0.1) with sizes of embryos recovered; correlation coefficients were -0.12 and -0.06, respectively.


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  		TABLE 2. Effect of in vitro culture with vitamin E on further growth of bovine embryos in vivo after transfer



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  		FIG. 1. Size distribution of embryos collected from recipients


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These studies were designed to determine whether the antioxidant vitamin E, with or without vitamin C (ascorbic acid) and/or EDTA, would protect in vitro-produced bovine embryos from oxidative damage in vitro in a 5% O2 atmosphere. Presumably it was more difficult, but more physiologically relevant, to show protective effects of antioxidants with 5% O2, 5% CO2, and 90% N2 than with 5% CO2 in air [16]. More embryos developed to expanded blastocysts when culture medium was supplemented with 100 µM vitamin E than with control treatment. Also, when transferred to recipients for 7 days, zygotes cultured to the morula stage grew into larger embryos in vivo (63% greater surface area). Furthermore, a higher percentage of embryos, albeit nonsignificant statistically, was recovered from the vitamin E group.

Vitamin E was first discovered as a dietary, lipid-soluble factor that could prevent fetal death and resorption in rats [23]. In other studies, vitamin E partially protected early murine embryos from the effects of heat shock [14], a cytotoxic event that likely is mediated by free radicals [24]. Although the rate of blastocyst development under heat shock conditions was not affected by culture with vitamin E ({alpha}-tocopheryl succinate), viability as determined by vital dye exclusion was improved [14]. Survival and development of rat conceptuses explanted on Day 8 of gestation were improved by culture with vitamin E [13]. These studies suggest a direct protective effect on the embryo in vitro, which may reflect a similar function in vivo.

The main biological function of vitamin E likely is to protect polyunsaturated fatty acids in membranes. Peroxidation of these membrane lipids can lead to structural damage, affecting function and permeability of membranes, eventually resulting in irreversible cell injury and death. A complex relationship exists between vitamin E and vitamin C, the major water-soluble antioxidant. In vitro, vitamin C regenerates vitamin E molecules that have undergone free radical attack and may also spare vitamin E from oxidation by reacting directly with free radicals [8]. We expected the combination of vitamins E and C in culture medium to improve conditions for embryonic development, more than with vitamin E alone, by elevating the antioxidant action of vitamin E. Instead, our results show that conditions worsened when vitamin C was added. A very similar observation was reported recently with human sperm incubated in vitro [25]; separately, vitamin E and vitamin C protected DNA from oxidative damage, but in combination damage was even higher than that in controls. Both vitamins are strong reducing agents and may act as prooxidants by maintaining iron and other metals in a reduced state, thus promoting lipid peroxidation [9]. Relative availability of free oxygen and metals reduced the half-life of vitamin C from weeks in the body to a few hours in culture, leading to production of oxidants in the media surrounding cultured cells [26]. Even with oxygen reduced to 5%, it is possible that vitamin C was acting as a prooxidant, not an antioxidant, in our culture system.

Adding EDTA to bind trace amounts of free transition metals was ineffective for enhancing the benefits of the antioxidant vitamins. Chelation of iron by EDTA restricts availability of ferrous ions. Lipid peroxidation requires both ferric and ferrous ions in biological systems [27]; therefore EDTA could theoretically inhibit lipid peroxidation under some embryo culture conditions. We did not measure lipid peroxidation directly in this study, but we would expect enhanced embryonic development if peroxidation were indeed prevented by EDTA. Other studies in this laboratory using culturally defined media, with and without EDTA, under similar conditions but without vitamin E, have demonstrated a benefit of including EDTA in the medium [16]. The interaction of vitamins E and C with EDTA may negate this effect, although dissecting the mechanisms of that interaction is beyond the scope of this study.

In summary, we have demonstrated that culture of bovine embryos with 100 µM vitamin E resulted in development of more embryos to early and expanded blastocysts, compared to embryos cultured in the control medium, and that this benefit was reversed when 100 µM vitamin C was also added. We have also shown that embryos treated in vitro with vitamin E were larger than control embryos after transfer and collection from recipient cows and heifers. This benefit was realized even under relatively low oxygen tension (5%) and with small amounts of albumin, which itself acts as an antioxidant [28]. Therefore this study provides evidence that vitamin E enhances bovine embryonic development in vitro, presumably by affording protection from reactive oxygen species.


   ACKNOWLEDGMENTS
 
We thank Anna Blaszczyk, Zella Brink, Richard Funston, Michael Holland, and Young-Gie Chung for technical assistance. LH and FSH were provided by the USDA and the National Hormone and Pituitary Program, NIDDK, NICHD, National Institutes of Health.


   FOOTNOTES
 
First decision: 15 February 1999.

1 This research was supported by USDA CGP 92-37203-7765 and the Colorado State University Experiment Station through Regional Project W-171. Back

2 Correspondence: G.E. Seidel, Jr., ARBL Building, Foothills Campus, Fort Collins, CO 80523. FAX: 970 491 3557; gseidel{at}cvmbs.colostate.edu Back

Accepted: September 8, 1999.

Received: January 11, 1999.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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