Theriogenology 61 (2004) 561572

Amino acid metabolism of preimplantation bovine embryos cultured with bovine serum albumin or polyvinyl alcohol
Nicolas M. Orsi1, Henry J. Leese*
Department of Biology, The University of York, PO Box 373, York YO10 5YW, UK Received 5 March 2003; received in revised form 3 June 2003; accepted 8 June 2003

Abstract Bovine serum albumin (BSA) is an embryotrophic macromolecule used in embryo culture media, which is commonly replaced with synthetic compounds, such as polyvinyl alcohol (PVA). This study compared the effect of BSA and PVA on the development, blastocyst cell number and amino acid metabolism of preimplantation bovine embryos in vitro. Embryos were produced by in vitro maturation and fertilization of immature oocytes from abattoir-derived ovaries. Zygotes were cultured in synthetic oviduct uid with either 4 mg/ml BSA (SOFaaBSA) or 1 mg/ml PVA (SOFaaPVA) in microdrops with a mineral oil overlay at 39 8C under a 5% O2/5% CO2/90% N2 atmosphere. Blastocyst rate and cell numbers were determined after 123 h of culture. In parallel, single expanding blastocysts grown in either medium were incubated in microdrops for 12 h. Amino acid prole of spent drops was determined by high performance liquid chromatography. Replacing BSA with PVA depressed blastocyst rate and cell numbers, and led to quantitative and qualitative differences in amino acid appearance, disappearance and turnover. These differences could partly be due to an increase in free intracellular amino acid concentration in SOFaaBSA embryos derived from hydrolysis of endocytosed BSA, and argue against the inclusion of PVA in bovine embryo culture media. # 2003 Elsevier Inc. All rights reserved.
Keywords: Bovine embryo; PVA; BSA; Amino acid metabolism

1. Introduction There is an accepted need for the inclusion of a macromolecule or macromolecule substitute in media for mammalian oocyte maturation, fertilization and early embryo
* Corresponding author. Tel.: 44-1904-328585; fax: 44-1904-432860. E-mail address: hjl1@york.ac.uk (H.J. Leese). 1 Present address: Academic Unit of Paediatrics, Obstetrics and Gynaecology, D Floor, Leeds General Infirmary, Belmont Grove, Leeds LS2 9NS, UK.

0093-691X/$ see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0093-691X(03)00206-1

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culture. Crystalline BSA was originally used by Whitten [1] and has since remained the most commonly used macromolecule, for the culture of embryos from the mouse [2,3], human [4,5] and domestic ruminants [6,7]. BSA improves embryo development, blastocyst formation and hatching rates in vitro. It may have a nutritional role by providing amino acids after hydrolysis [8,9], thereby maintaining intracellular amino acid pools [10]. BSA may also provide as yet undened embryotrophic compounds (e.g. citrate, steroids) [8,1114], function as a heavy metal ion chelator/free radical scavenger, protect cellular constituents against the effect of toxins, and regulate redox potential, pH and osmolarity [8,1518]. Furthermore, BSA is essential for capacitation [19] and the acrosome reaction in vitro [20,21]. The need to replace BSA with a synthetic, non-metabolisable substitute arose in experiments investigating the role and metabolism of exogenous amino acids in early embryos [14,16,22,23]. Concerns associated with the transmission of pathogens and the move to fully dened media in many species have contributed to a decline in the use of albumin in favor of synthetic macromolecules [2426]. Polyvinylpyrrolidone (PVP) was initially identied as a candidate macromolecule [16], but was eventually replaced by polyvinyl alcohol (PVA) [21], because of its toxicity [27]. The purity of BSA preparations can determine developmental outcomes, as reported for hamster zygotes [28]. Even higher-grade albumin preparations retain small, undened molecules after purication [8,14], which likely account for the variability in the embryotrophic effect observed with different batches of BSA [2933]. Cattle embryos grown with different batches of BSA display altered levels of developmentally important gene transcripts [34]. Although de novo protein synthetic rates do not differ between embryos cultured with BSA and PVA [35], trophectodermal uptake of exogenous protein by endocytosis, a phenomenon described in the mouse [36], sheep [37] and cow [10], may account for the lower protein content in PVA cultured embryos [10]. This endocytic activity appears to be regulated by extracellular protein availability [36]. Whether there are benets in using macromolecules other than BSA is a subject of debate. Although some authors report that zygotes and two-cell mouse embryos require a xed nitrogen source (e.g. BSA, amino acids or glutathione) [8,16,37], others have shown that zygotes [8,38,17], two-cell [39], and eight-cell [40] murine embryos can develop into blastocysts without BSA provided PVA or EDTA are present in the medium. Other studies support an intermediate view: glutamine alone, for instance, appears to be sufcient to support murine preimplantation development in the presence of PVA [8]. Likewise, eightcell hamster embryos form blastocysts in the absence of BSA, provided amino acids and vitamins are present [26]. This pattern is similar in the rabbit, where the benets of BSA supplementation are determined by the nature of the base medium [41], although a mixture of amino acids is required in order to complete preimplantation development [42]. In cattle embryos, substitution of BSA with PVA adversely affects development [34,35,43,44], hatching rate, blastocyst cell number [44] and viability after vitrication [46]. This is associated with an increase in pyruvate consumption and a decrease in oxygen consumption, indicating a non-oxidative fate for this substrate [43]. Bovine embryo development can occur at sustained rates even in the presence of PVA, if the base medium is supplemented with myo-inositol and citrate [8,41].

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The desirability of replacing the BSA in culture media with PVA in an endeavor to create dened conditions remains open to question, even for the purposes of metabolic assessment. Culture of embryos with PVA isolates embryos from the major physiological macromolecule of maternal tract uid (i.e. BSA) [45], whose function extends beyond that of providing nutrients. Little is known about the biological effects of PVA. It is likely to be taken up by endocytosis and may have direct effects on the embryo which may not be apparent until after transfer. It is surprising that little interest has focused on the effects of PVA on embryo amino acid metabolism, since culture in PVA-containing medium alters embryo carbohydrate metabolism [46]. The present study used in vitro-produced bovine embryos to determine whether replacing BSA with a synthetic macromolecule (PVA) inuences the metabolism of a physiological mixture of amino acids.

2. Materials and methods 2.1. Embryo production and culture Cattle embryos were generated by in vitro maturation and in vitro fertilization of immature oocytes aspirated from abattoir-derived ovaries, as previously described [47,48]. Briey, oocytecumulus complexes were matured for 14 h in tissue culture medium 199 supplemented with 10% fetal calf serum, 1 mg/ml oestradiol, 10.9 ng/ml broblast growth factor, 0.47 mg/ml epidermal growth factor, 0.025 IU luteinising hormone (Ferring Pharmaceuticals, Berkshire, UK), 0.025 IU follicle-stimulating hormone (Ferring Pharmaceuticals), 24 mg/ml apo-transferrin and 1 mM pyruvate. Fertilization was performed in fertilization Tyrodes albumin pyruvate lactate medium (Fert TALP) with 1 106 sperm/ml of freeze-thawed semen from a sire of proven fertility. All chemicals were purchased from Sigma, Poole, Dorset, UK, unless otherwise specied. Two cohorts of presumptive zygotes were grown in parallel, in groups of 14/20 ml microdrops SOFaaBSA (n 12) and SOFaaPVA (n 13) with a mineral oil overlay at 39 8C under a 5% O2/5% CO2/90% N2 atmosphere. In SOFaaPVA, the BSA component of the medium was replaced by 1 mg/ml PVA. Zygotes cultured in this medium were washed four times prior to culture to ensure minimal carry over of BSA. Blastocyst rate was recorded after 123 h of culture. 2.2. Evaluation of blastocyst cell numbers Cell counts were performed on expanded bovine blastocysts grown in SOFaaBSA (n 17) and SOFaaPVA (n 8) after 123 h of culture, as previously described [49,50]. The zona pellucida was removed by exposure to acid Tyrodes solution; the blastocysts were then transferred to absolute ethanol supplemented with 50 mg/ml bisbenzimide for 48 h, after which they were transferred to 2 ml glycerol drops on siliconised slides, immobilized with a cover slip, and labeled nuclei counted under UVon a Leica microscope (Milton Keynes, UK).

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2.3. Amino acid incubations and analyses Expanding bovine blastocysts cultured in both media were selected on days 7 and 8 of culture in order to remove the bias towards faster development in SOFaaBSA (personal observation). Embryos were preincubated for 30 min in 500 ml of either SOFaaBSA or SOFaaPVA to allow equilibration of intracellular amino acid pools with the medium. They were then washed twice in SOFaaBSA (n 75) or SOFaaPVA (n 86), and incubated singly for 12 h in 1 ml of either medium with a mineral oil overlay, as described for culture. Blastocysts were discarded and the spent drops diluted 1:40 with high performance liquid chromatography (HPLC)-grade water, processed and analyzed for amino acid concentrations by HPLC, as previously described [22,23], using o-phthaldialdehyde as a pre-column derivatizing reagent. Amino acids were analyzed on a Kontron 500 series automated HPLC system tted with a Jasco uorescence detector and a 5 mm diameter, 4:5 mm 250 mm Hypersil ODS-16 column and guard column (Jones Chromatography, Hengoed, Mid Glamorgan, UK). 10 mM D-a-amino butyric acid (DABA), a non-metabolisable amino acid, was included in all medium formulations used in this study as an internal standard against which to correct any potential errors in sample dilution. Solvent A was 80% sodium acetate pH 5.9, 20% methanol, to which 1214% tetrahydrofuran was added; solvent B was 80% methanol and 20% sodium acetate. The elution gradient was 100:0 solvent A:solvent B, decreasing to 86:14 solvent A:solvent B at 12 min, 50:50 solvent A:solvent B at 24 min and 0:100 solvent A:solvent B at 40 min. Flow rate was maintained at 1.2 ml/min. 2.4. Data presentation and statistical analysis Zygote to blastocyst rates were expressed as a percent and were arcsine log transformed for the purposes of statistical analysis. Amino acid proles (disappearance from, and appearance in, the medium) were expressed as pmol per embryo per hour and the data permuted to yield overall appearance (the sum of all amino acids appearing in the medium); overall disappearance, and turnover (the sum of overall appearance and disappearance). Turnover provides an estimate of overall embryo amino acid metabolic activity. The variation in individual amino acid proles has been accounted for by using the expression percent of turnover, which corrects for differences in metabolic rate, cell number, etc. between individual blastocysts. Nitrogen appearance, disappearance and turnover were evaluated based on the number of atoms of nitrogen present in each of the amino acid structures. All data were presented S.E.M. Differences between groups were tested by Students t-test or, in the case of those characterized by non-parametric distributions upon transformation (as assessed by AndersonDarling tests), by MannWhitney U (Wilcoxons signed rank) tests.

3. Results A greater proportion of embryos cultured in SOFaaBSA developed to the blastocyst stage. Furthermore, these had a greater number of cells than their SOFaaPVA counterparts (P < 0:05; Table 1).

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Table 1 Day 8 blastocyst rate and cell number of bovine embryos cultusred in either SOFaaBSA or SOFaaPVA (different superscripts indicate statistically significant differences between groups; P < 0:05) Culture group SOFaaBSA SOFaaPVA Overall cleavage rate (%) 68.83 (3.36) 68.45 (5.03) Day 8 blastocyst rate (%) 21.98 (0.41) 18.33 (0.46)b
a

Blastocyst cell number 105.33 (3.93) 89.83 (4.20)

Blastocysts derived from embryos cultured in SOFaaBSA had a signicantly different appearance/disappearance of aspartate, glutamate, asparagine, glycine, arginine, alanine, tryptophan, isoleucine and lysine compared to their SOFaaPVA counterparts (Fig. 1). However, no signicant differences in overall amino acid appearance and disappearance were recorded between blastocysts grown in SOFaaBSA and SOFaaPVA (Fig. 2). By contrast, a signicant difference was recorded in amino acid turnover (26:98 1:08 and 24:15 0:93) but not net ux (the difference between overall appearance and disappearance; 0:01 1:05 and 0:37 1:07) between blastocysts cultured in SOFaaBSA and SOFaaPVA, respectively (P < 0:05). The data (Fig. 3) indicated that blastocysts derived from embryos cultured in SOFaaBSA had a signicantly different proportional appearance/disappearance of aspartate, asparagine, arginine, alanine, valine, isoleucine and leucine compared to their SOFaaPVA counterparts. No signicant differences were recorded between appearance (49:44 1:98 and 48:85 2:06), disappearance (51:11 2:13 and 51:15 2:06) and net ux (23:32 2:17 and
8 Appearance/disappearance (pmol/embryo/h)

***
6 4 2 0
R TR P M ET V A L PH E LU A SN TH TY

SOFaaBSA SOFaaPVA

*** ***
SE R SP IS LN LY R RG LA

**
A

-2 -4

*** **

**

**

**

-6
Fig. 1. Individual amino acid appearance and disappearance for single bovine blastocysts cultured in either SOFaaBSA or SOFaaPVA (P < 0:01; P < 0:001).

LE

U LY S

IL E

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Appearance/disappearance (pmol/embryo/h)

20 15 10 5 0 -5 -10 -15 -20

SOFaaBSA SOFaaPVA Appearance Disappearance

Fig. 2. Overall amino acid appearance and disappearance with single blastocysts cultured in either SOFaaBSA or SOFaaPVA.

30:44 2:46) in SOFaaBSA and SOFaaPVA embryos, respectively, when the data were expressed as a percent of turnover. Signicant differences in nitrogen appearance and disappearance by embryos in SOFaaBSA and SOFaaPVA were recorded for aspartate, glutamine, asparagine, glycine, arginine, alanine, tryptophan, isoleucine and lysine (Fig. 4). A signicant difference in nitrogen disappearance but not appearance was recorded between blastocysts derived from embryos cultured in SOFaaBSA or SOFaaPVA (Fig. 5). A signicant difference in nitrogen turnover (43:14 1:85 and 37:40 1:44) but not net ux (5:36 1:81 and 2:65 1:52) was recorded between blastocysts derived from embryos cultured in SOFaaBSA and SOFaaPVA, respectively (P < 0:05).

30 Appearance/disappearance (% of turnover)
SOFaaBSA

25 20 *** 15 10 ***

***

SOFaaPVA

*** **
***

5 0
ASP GLU

ASN

SER

HIS

GLN

GLY

THR ARG ALA TYR

TRP

MET VAL

PHE

ILE

LEU

LYS

Fig. 3. Individual amino acid metabolism, expressed as a percent of turnover, by single blastocysts cultured in either SOFaaBSA or SOFaaPVA (P < 0:05; P < 0:01; P < 0:001).

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567

Nitrogen appearance/disappearance (N atoms/embryo/h)

SOFaaBSA SOFaaPVA
5

*** ***

***
0 ASP GLU

*
ASN SER HIS GLN

***
GLY THR ARG ALA TYR

*
TRP MET VAL PHE ILE LEU LYS

**
-5

-10

***
-15

Fig. 4. Nitrogen appearance and disappearance on an individual amino acid basis by single blastocysts cultured in either SOFaaBSA or SOFaaPVA (P < 0:05; P < 0:01; P < 0:001).

Appearance/disappearance (pmol/embryo/h)

20 15 10 5 0 -5 -10 -15 -20 SOFaaBSA SOFaaPVA Appearance Disappearance

Fig. 5. Overall nitrogen appearance and disappearance for single blastocysts cultured in either SOFaaBSA or SOFaaPVA (P < 0:05).

4. Discussion Culture of bovine embryos in SOFaaPVA did not support the developmental rates recorded with BSA, even in the presence of amino acids, in agreement with previous reports [34,35,43,44,51]. The PVA-induced reduction in cavitation and hatching rates was associated with reduced blastocyst cell numbers, as reported in some [44], but not other [43] studies. This suggests that embryos grown in PVA may have reduced viability. Although calves have been obtained from embryos cultured with PVA, the medium in these experiments was supplemented with citrate and myo-inositol; two compounds that restore developmental capacity to BSA-supplemented levels [52].

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Table 2 Amino acid composition of BSA (adapted from Hilger et al. [54]) Amino acid Leucine Lysine Glutamate Alanine Aspartate Valine Cysteine Threonine Proline Serine Phenylalanine Arginine Glutamine Tyrosine Histidine Glycine Asparagine Isoleucine Methionine Tryptophan Number of residues (%) 61 59 59 47 40 36 35 33 28 28 27 23 20 20 17 16 14 14 4 2 (10.46) (10.12) (10.12) (8.06) (6.86) (6.17) (6.00) (5.66) (4.80) (4.80) (4.63) (3.95) (3.43) (3.43) (2.92) (2.74) (2.40) (2.40) (0.67) (0.34)

The present study indicates that the replacement of protein in the culture medium with PVA leads to differences in amino acid metabolism. One explanation for these differences involves taking into account the amino acid composition of BSA (Table 2). Following endocytosis [10], BSA may be hydrolyzed, thereby most likely increasing leucine, lysine and glutamate intracellular pools. Pemble and Kaye [53] reported that murine blastocysts internalize BSA-containing extracellular uid through endocytosis at a rate equivalent to their total volume per hour. If this occurs in the bovine blastocyst (early-expanded volume: 1.53.6 nl; N. Gopichandran, personal communication) incubated in SOFaaBSA (4 mg/ml BSA), albumin uptake can be calculated as 6.014.4 ng/h BSA. Assuming a MW of 250,000 for BSA, this equates to 0.0240.0576 pmol/h BSA and, assuming 583 residues in the protein, a potential amino acid provision of 13.9933.58 pmol/h amino acids. As discussed by Pemble and Kaye [53], this is likely to represent a signicant contribution to the embryonic pool of xed nitrogen. In agreement with this proposition, there was a net appearance of lysine for embryos cultured in SOFaaBSA, whilst their SOFaaPVA counterparts showed a net consumption. Lysine is an essential amino acid that cannot be synthesized from its corresponding a-ketoacid. Glutamate was consumed (SOFaaBSA) or produced (SOFaaPVA). Increased intracellular glutamate derived from endocytosed BSA may have been converted to glutamine, which appeared in the medium, by the glutamine synthetase reaction, which xes NH4 . Increased ammonium production would be consistent with the increased rate of protein degradation resulting from BSA endocytosis. The present data suggest that differences in amino acid availability result in alterations in their appearance/disappearance from the medium, in agreement with previous reports [55]. BSA hydrolysis will alter individual intracellular amino acid concentrations and may

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affect energy-generating pathways in the embryo (e.g. carbohydrate metabolism) [43]. Thus, the present results suggest that BSA may affect preimplantation metabolism, either directly through the provision of amino acid substrates, or indirectly through the action of bound embryotrophic compounds; for example: steroids, myo-inositol, growth factors, other proteins/enzymes and/or enzyme cofactors [14]. Alanine appearance was greater in embryos cultured in SOFaaBSA (Figs. 1 and 3). Alanine may have been derived from the transamination of pyruvate as a means of xing ammonium [23,56]. This is not surprising since a greater rate of amino acid deamination in SOFaaBSA embryos will be a consequence of BSA endocytosis and hydrolysis. It is likely that BSA-derived amino acids were either incorporated into protein or oxidised (assuming no BSA storage). Eckert et al. [43] demonstrated that embryos cultured in SOFaaBSA consumed more oxygen than their SOFaaPVA counterparts, despite having a signicantly lower pyruvate uptake. The extra oxygen consumed was unlikely to have participated in glucose oxidation, as the glycolytic index (percent glucose disappearance accountable as lactate appearance) of both groups was virtually identical. More likely, the extra oxygen may have participated in the oxidation of the amino acid carbon skeletons after deamination [57]. The present amino acid proles obtained for SOFaaBSA and SOFaaPVA were similar to those obtained in previous reports [23,55,58], although direct comparison is made difcult by variations in methodology and species studied [22,23,51,58,59]. A recent study comparing the amino acid metabolism of SOFaaPVA and SOFaaBSA bovine blastocysts has demonstrated that the amino acid prole of these two groups only differed with respect to alanine production, which was lower in SOFaaPVA [51]. Although this nding agrees with the present data, the study did not identify any other differences in amino acid prole, possibly due to the small sample size used. The lower amino acid turnover recorded in SOFaaPVA embryos was indicative of depressed amino acid metabolism. This may be a marker of reduced viability, as blastocyst development and cell numbers were reduced in the presence of PVA. Differences in nitrogen appearance and disappearance demonstrated that most of the amino nitrogen consumed was attributable to aspartate, glutamine and lysine (for SOFaaBSA only), whilst the majority of the recorded loss occurred through alanine. Unaccounted nitrogen was probably principally lost as NH4 , at a rate of around 4 pmol per embryo per hour, since bovine embryos do not have a functional urea cycle [60]. In conclusion, our data support the view that exogenous BSA (to which embryos are exposed in the female reproductive tract) is endocytosed, especially at the blastocyst stage. We propose that, following intracellular hydrolysis, its constituent amino acids contribute to the nitrogen metabolism of the embryo. Embryos cultured in PVA exhibited different amino acid proles than those grown with BSA, as well as showing depressed developmental rates and cell numbers. Taken together, these observations argue against the use of PVA in bovine embryo culture media. Acknowledgements The authors gratefully acknowledge excellent technical support from Mr. Peter Humpherson, and nancial support from a University of York Studentship and the European Commission.

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