THE JOURNAL OF EXPERIMENTAL ZOOLOGY 257:350-359 (1991)

Culture Medium and Protein Supplementation Influence In Vitro Fertilization and Embryo Development in the Domestic Cat L.A. JOHNSTON, A.M. DONOGHUE, S.J. O’BRIEN, AND D.E. WILDT National Zoological Park, Smithsonian Institution, Washington, District of Columbia 20008 (L.A.J., A.M.D., D.E.W.); Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21 701 (L.A.J., S.J. O ~ B J

ABSTRACT The influence of culture medium and protein supplements on in vitro fertilization (IVF) and morula-to-blastocyst development in culture was examined in the domestic cat. In Study I, follicular oocytes were fertilized and cultured in 1)modified Krebs Ringer bicarbonate (mKRB); 2) modified Tyrode’s solution (TALP) without phosphate or glucose; or 3) Ham’s F10. All media contained bovine serum albumin (BSA). Fertilization rates were similar (P > .05) among mKRB (75.0%),TALP (70.6%), and Ham’s F10 (80.0%)treatments. Compared to TALP (77.8%),more (P < .05) embryos in Ham’s F10 (95.0%) developed to the morula stage; development of mKRB embryos (88.9%)was intermediate and not different (P > .05). Study I1 evaluated the effects of protein availability and type on IVF and embryo growth. Ham’s F10 was supplemented with polyvinylalcoho1 (PVA, 2 mg/ml), BSA (4mg/ml), fetal calf serum (FCS, 5%), or estrous cat serum (ECS, 5%) and used to fertilize and culture embryos. Fertilization was enhanced (P < .05) using either FCS (84.0%) or ECS (85.2%) compared to PVA (67.3%). Oocytes exposed to BSA fertilized at a rate (76.1%) similar (P > .05) to other treatments. Embryos exposed to all four treatment groups (PVA, 82.8%; BSA, 82.8%; FCS, 92.9%; o r ECS, 97.8%) were equally capable (P > .05) of becoming morulae. However, more FCS- or ECS-supplemented morulae continued to the early blastocyst stage (30.8%, 22.276, respectively, P < .05) than PVA- (10.3%)or BSA- (13.8%) exposed morulae. In the domestic cat IVF system, the type of medium and protein supplement used appears to have a greater impact on embryo development in vitro than on fertilization. There also is a morula-toblastocyst developmental block for cat embryos resulting from IVF which is partially circumvented by adding serum to the culture medium. Ham’s F10 containing FCS or ECS produces the highest incidence of fertilization (-85%), and -2-fold more of these embryos develop in culture to blastocysts compared to embryos supplemented with PVA or BSA.

Although in vitro fertilization (IVF) can be achieved routinely in the domestic cat, no data are available concerning the optimal culture requirements for fertilization and early embryo development in this or any carnivore species (Mahi and Yanagimachi, ’76; Goodrowe et a]., ’88;Johnston et al., ’89).Our overall understanding of the regulatory mechanisms for mammalian fertilization and early embryogenesis is based largely on mouse, rabbit, and hamster studies and, t o a lesser extent, on examination of cattle, sheep, and pig embryos. From these studies, there is ample evidence that ova and embryos from different taxa vary in fertilization and culture requirements and appear to rely on different energy and protein sources (see review, Bavister, ’87a,b; Kane, ’87a). There are two types of culture media for IVF 0 1991 WILEY-LISS, INC.

and embryo culture: 1)“simple” media which consist of balanced salt solutions with protein and energy substrates; and 2) “complex”media which contain these components as well as vitamins, amino acids, and trace minerals. Several medium components influence embryo development in. vitro including glucose, phosphate, amino acids (including glutamine), and lactate, but empirical studies have been conducted using only a few species (pig, Davis and Day, ’78; Meyen et al., ’89; rabbit, Kane, ’87b; hamster, Schini and Bavister, ’88;Seshagiri and Bavister, ’89a,b;mouse, Chatot et al., ’89).In addition, a macromolecular compoReceived March 2, 1990; revision accepted August 6, 1990. Address reprint requests to Dr. David Wildt, National Zoological Park, Smithsonian Institution, Washington, DC 20008. Dr. L.A. Johnston’s present address: Henry Doorly Zoo, Omaha, NE 68107.

CULTURE OF CAT EMBRYOS

nent often is present in the culture medium t o enhance mammalian embryo development (Cholewa and Whitten, '70; Bavister, '87a). Although the usefulness of exogenous protein in the culture of certain species of embryos is controversial, conflicting experimental evidence probably is due t o inherent variabilities among different sera and bovine serum albumin (BSA) preparations (Kane, '87a,b). There are at least four reasons for sustaining IVF-derived felid embryos in culture until later developmental stages. First, such information can serve as a database for developing optimal in vitro culture systems for the Carnivora order which has received little research attention. Second, access t o later stage embryos would facilitate transfers of embryos directly into the uterus, perhaps by atraumatic approaches like laparoscopy (Schiewe et al., '84). Third, successful embryo cryopreservation may require later stage embryos because, in some species, embryos containing eight blastomeres or fewer have reduced post-thaw viability (Wilmut, '86). Successful cryopreservation of in vivo-derived morula stage, domestic cat embryos has been demonstrated recently (Dresser et al., '88). Fourth, the domestic cat serves as a useful experimental model for reproductive studies of nondomestic felids (Wildt, '90, '91). Understanding the general nutritive and energy requirements for early stage domestic cat embryos will provide the foundation for comparative developmental studies of nondomestic felid species. Two studies were conducted to begin defining the optimal culture requirements for domestic cat preimplantation embryos produced by IVF. Simple media were tested in an attempt t o identify specific chemical prerequisites for fertilization and embryo development. However, a complex medium also was evaluated because our ultimate objective is to provide a system that supports in vitro survival for embryos from an array of nondomestic felid species. The three media tested were modified Krebs Ringer bicarbonate (mKRB), Tyrode's solution (TALP), and Ham's F10. Developed by Toyoda and Chang ('741, mKRB contains sodium lactate, sodium pyruvate, glucose, and no amino acids or vitamins. Modified Tyrode's solution (TALP), formulated by Bavister and Yanagimachi ('77), is considered a "simple" medium, containing sodium lactate and sodium pyruvate. TALP has been used to culture hamster, cow, and rhesus monkey embryos successfully (Bavister, '87b). In our study, TALP, modified fur-

351

ther to eliminate glucose and phosphate, was tested because it has been used t o culture hamster embryos successfully beyond the four-cell stage developmental block (Schini and Bavister, '88). Ham's F10, a complex medium originally designed t o reduce the need for serum supplements in diploid cell line cultures, contains pyruvate, glucose, amino acids (including glutamine), and vitamins. Our specific aim was t o determine the influence of culture medium and protein availability and source on the incidence of IVF and embryo development of domestic cat follicular oocytes.

MATERIALS AND METHODS Animals and animal maintenance Random-source, adult female cats (n = 28) were housed according to earlier detailed descriptions (Goodrowe et al., '88; Howard et al., '90) and provided a commercial feline diet (Purina Cat Chow, Ralston Purina, St. Louis, MO) and water ad libitum. The single male sperm donor was housed in a separate cage and provided the same diet. Twelve hours of artificial fluorescent illumination were provided daily. Induction of ovarian activity and oocgte recovery Females were anesthetized and subjected to a pre-gonadotropin treatment, laparoscopic examination of ovarian activity using the detailed descriptions of Goodrowe et al. ('88).Adult, domestic cats with no follicular development or luteal tissue on either ovary were given 150 IU pregnant mares' serum gonadotropin (PMSG; i.m., Sigma Chemical Co., St. Louis, MO) to stimulate ovarian follicular development followed 84 h later with 100 IU human chorionic gonadotropin (hCG; i.m., Sigma Chemical Co.) to induce oocyte maturation (Miller et al., '89). Twenty-four to 27 h post-hCG, oocytes were recovered laparoscopically from ovarian follicles ( 2 2 mm in diameter) (Goodrowe et al., '88). Follicle contents were aspirated into collection tubes containing either mKRB (Toyoda and Chang, '74; Niwa et al., '85) (Study I) or Ham's F10 (Study 11)containing 40 units of heparidml. Collection tubes were maintained at 3537°C during and after the aspiration procedure. The contents of each tube were emptied into a 60 x 15 mm plastic Petri dish and rinsed with 5 ml of warm equilibrated medium. The dishes were searched for oocytes which were transferred t o fresh medium and placed in a 5% C02 in air, humidified chamber at 37°C. Each oocyte and its

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L.A. JOHNSTON ET AL.

surrounding cumulus cell mass were assessed for maturational status based on morphological criteria developed for domestic cat oocytes (Goodrowe et al., '88; Johnston et al., '89). Only oocytes designated as mature by expansion of the corona radiata and cumulus cell mass were used. Mature oocytes were washed three times in medium under oil, placed in fresh medium, and returned to the incubator.

Collection and processing of spermatozoa To control for potential male effects on fertilization and embryo development (Fukui et al., '88; Eyestone et al., '89a), only a single proven breeder male was used as a sperm donor. Spermatozoa were collected using a n electroejaculation technique described for the domestic cat (Wildt et al., '83). Electroejaculated semen was subjected t o swim-up processing (Goodrowe et al., '88; Howard et al., '90). In brief, this involved transferring electroejaculated semen into a 1.5 ml conical tube (Sarstedt Inc., Princeton, NJ), diluting with a n equal volume of medium, and then centrifuging for 8 min at 300g. The supernatant was aspirated and discarded and 150 pl of medium (mKRB, Study I; Ham's F10, Study 11)was layered slowly onto the resulting pellet and the sperm allowed a 1 h swim-up at room temperature. The layered aliquot was aspirated and assessed objectively for sperm concentration and subjectively for percent motility and progressive status (0 = no forward movement of spermatozoa to 5 = rapid, linear, forward movement) (Wildt et al., '83; Howard et al., '90). Only swim-up aliquots containing spermatozoa with a 75% motility and 3.0 progressive status rating were used for fertilization attempts. Spermatozoa were diluted to a final concentration of 2 x lo5 sperm celldm1 using the appropriate medium/protein supplement according to either the Study I or Study I1 protocol. In vitro fertilization and embryo culture Mature oocytes were co-cultured with processed spermatozoa (six-12 oocytes/insemination drop) at 37°C in 5% COz, 95%air atmosphere. After 12-18 h of culture, oocytes were removed from fertilization dishes and washed in a 0.2% hyaluronidase solution (Type 1-S from bovine testis, Sigma Chemical Co.) to remove cumulus cells and loosely attached spermatozoa. Oocytes were returned to culture in 100 pl drops of fresh medium, overlaid with oil, and co-cultured for an additional 12-18 h before assessing fertilization. Fertilized oocytes were those which cleaved to

TABLE 1. Formulation of media used for in vitro fertilization and culture of cat embryos Comnonent

mKRB

NaCl KC1 CaC1, KHZP04 MgS04 NaHC03 Na lactate Na pyruvate Glucose Amino acids Glutamine Vitamins

94.60 4.78 1.71 1.19 1.19 25.07 21.58 0.50 5.56

Concentration (mMi TALP Ham's F10 98.00 3.20 2.00 -

0.50 25.00 10.00 0.50 -

-

-

-

-

-

-

127.58 3.82 0.30 0.61 0.62 14.29 -

1.00 6.10

+ + +

the two-cell stage of development within 30 h of being placed in culture. Cleaved embryos were monitored for quality and stage of development at 24 h intervals for 7 days. Embryos considered viable were those which: 1) were symmetrical (or only slightly asymmetrical); 2) contained blastomeres which were spherical in shape and uniformly dark; and 3) demonstrated progressive development over time (increased number of blastomeres). Embryos were considered t o be degenerate if blastomeres were asymmetrical, pale, or lyzed and the embryo had arrested in growth while being maintained in vitro. Embryos with 2 3 2 cells were considered morulae, whereas embryos with a developing blastocoelic cavity were classified as early blastocysts. All embryos were stained using a DNA-specific Hoechst stain (Goodrowe et al., '88; Johnston et al., '89), and nuclei were counted.

Influence of culture medium on IVF and preimplantation embryo development Study I Three culture media were examined: 1)mKRB (water from Irvine Scientific, Santa Ana, CA; chemicals from Sigma Chemical Co.); 2) TALP (Bavister and Yanagimachi, '77) prepared without phosphate and glucose (Schini and Bavister, '88) (water from Irvine Scientific and chemicals from Sigma Chemical Co.); and 3) Ham's F10 (Irvine Scientific) (Table 1).Each medium was supplemented with BSA (4 mg/ml; Miles Scientific, Lisle, IL) and equilibrated at 37°C with 5% COz. A single electroejaculate, initially processed with mKRB, was aliquoted and diluted (1:100 dilution of processed semen to medium) with each me-

CULTURE OF CAT EMBRYOS

dium. Each experiment was repeated four times and media were prepared fresh on four different occasions over a period of 2 months. Oocytes from individual females were divided equally and randomly among the three test media (mKRB, n = 48; TALP, n = 51; Ham’s F10, n = 50) and then co-cultured with sperm previously diluted with the conspecific medium. Influence of protein on IVF and preimplantation embryo development Study I1 Based on the findings from Study I (see “Results”), Ham’s F10 was chosen as the optimal culture medium for Study 11.Using this complex medium, the effect of presence and type of protein on in vitro fertilization and embryonic growth was evaluated. Ham’s F10 was supplemented with polyvinylalcohol (PVA; 2 mg/ml; a no protein control, Sigma Chemical Co.), BSA (4 mg/ml; Miles Scientific), fetal calf serum (FCS; 5%; Gibco Laboratories, Grand Isle, NY), or estrous cat serum (ECS; 5%).For the latter, serum was collected and pooled from four female cats exhibiting behavioral estrus. FCS and ECS were heat-inactivated at 56°C for 30 min and filtered (Corning Filter Unit, 0.22 pm) before use. Electroejaculated spermatozoa, swim-up processed in Ham’s FlO supplemented with BSA (4 mg/ml), were aliquoted and diluted ( 1 : l O O as described above) with each treatment medium. Each experiment was repeated four times and media were prepared fresh on four different occasions over a period of 2 months. Follicular oocytes from individual females were distributed equally and randomly among each of the four culture treatments (PVA, n = 52; BSA, n = 46; FCS, n = 50; ECS, n = 54). Statistical analysis The proportion of oocytes fertilizing after coculture with spermatozoa and the proportion of developing embryos at a specific stage are expressed as percentages. Differences in fertilization and embryo development among treatment groups were evaluated by chi square (X2) analysis. The development (or growth) was scored as the proportion of embryos that reached the next progressive developmental stage after 24 (twocell), 48 (four- to eight-cell), 72 (>8- to 16-cell), 96 (>16- to 32-cell), and 120 (early blastocyst) h of culture. Embryo growth between treatments at a given examination time were evaluated by Tukey’s HSD procedure.

353

RESULTS Study I Medium had no effect (P> .05) on in vitro fertilization success; cleavage rate for cultured oocytes ranged from 70.6% (36/51 oocytes) for TALP t o 80.0% (40/50 oocytes) for Ham’s F10 with mKRB intermediate a t 75.0% (36/48 oocytes). However, medium did influence (P < .05) the proportion of embryos developing in culture. More embryos advanced t o the morula stage with Ham’s F10 (95.0%) compared to TALP (77.8%); the proportion of mKRB-cultured embryos (88.9%) was intermediate and not different (P > .05) (Fig. 1). From 2.6% (Ham’s F10) to 7.1% (TALP) of all morulae advanced to the blastocyst stage in vitro, and this development was unrelated (P > .05) t o medium (Fig. 1). In vitro embryo growth was similar (P> .05) between mKRB and Ham’s F10 test media. The proportion of all embryos at the expected stage of development at a given examination time was not different between these two media (Fig. 2). However, both media enhanced (P < .05) development compared to that achieved with TALP (Fig. 2). Study 11 Fertilization rate was increased (P < .05) by using either FCS or ECS as protein supplements compared to the PVA control (Table 2). Oocytes exposed to BSA fertilized at an intermediate rate which was not different (P> .05) from other treatments (Table 2). Embryos from each treatment group were equally capable of developing to the morula stage (range, PVA and BSA, 82.8% to ECS, 97.8%) (Fig. 3). Culturing in either FCS(30.8%) or ECS- (22.2%) supplemented medium resulted in twice as many morulae developing t o the blastocyst stage compared t o either PVA (10.3%) or BSA (13.8%)treatments (Fig. 3). Furthermore, embryos cultured in either FCS or ECS had greater (P < .05) development at each time point examined compared to those maintained in either PVA or BSA (Fig. 4). However, each of the three protein sources (BSA, FCS, ECS) stimulated greater embryo growth (P < .05) in vitro compared t o the nonprotein PVA control (Fig. 4).

DISCUSSION In the domestic cat IVF system described here, higher rates of IVF (>70%) were achieved than reported earlier by Goodrowe et al. (’88) of our laboratory. We previously demonstrated that IVF in cats is sensitive to hCG dosage and especially

354

L.A. JOHNSTON ET AL. 100

1 32/36

38/40 a

'' 28/36

80

60

Morula Blastocyst

40

20

0

l--

mKRB

1

TALP

Ham's F10

Medium Fig. 1. Percentage of embryos developing to morulae or blastocysts at 96 h of culture in three different media. Numbers above bars indicate total morulaeitwo-cell embryos or total early blastocystsimorulae. a.bTalueswithin treatments with different superscripts differ (P < .05).

2-cells

80

Y

E

-

a

4 to

28 to

%cells

16-cells

a

>16 to 32-cells a

a

early blastocysts a

a

60 -

5

40

-

0

U

e W u L

i

mKRB TALP Ham'sF

20 -

0'

T

24

-7

48

T

-4-

72

96

120

Hours in Culture Fig. 2. Embryo development after 24, 48, 72, 96, and 120 h of culture in three different media. a.bValueswithin treatments with different superscripts differ (P< .05).

CULTURE OF CAT EMBRYOS

355

used, then high rates (>70%) of embryo cleavage t o the morula stage can be expected. Culture medium also had a major impact on the No. of No. of Protein oocytes oocytes fertilized (%) ability of newly cleaved embryos to advance to later stages of preimplantation development. Our PVA 52 35 (67.3)l 35 (76.1)l~' laboratory has used mKRB for routine IVF and BSA 46 culture of domestic cat (Goodrowe et al., '881, 42 (84.0)' FCS 50 54 46 (85.2)' ECS leopard cat, Felis bengalensis (Goodrowe et al., 'SS), and puma, Felis concolor (Miller et al., '901, l,'Values within treatments with different superscripts differ (P < ,051. embryos with satisfactory results. However, longterm culture of domestic cat embryos did not result in blastocoel development. Bowen ('77) proto the PMSG-to-hCG interval (Goodrowe et al., duced domestic cat embryos by IVF using ductus '88). Most recently, Miller et al. ('89) demon- deferens spermatozoa, ovulated ova, and modified strated higher IVF rates when the PMSG-hCG Ham's F10 containing BSA and found that 6.5% interval was 84 (62.5%), 88 (58.2%),or 92 (55.7%) (3146) of embryos developed into blastocysts. Modh compared t o 80 (48.3%)and 96 (40.7%) h. This ified Ham's F10 differs primarily from Ham's F10 finding appears related to intrafollicular oocyte (used in our study) in that glucose is absent in the maturation which is gonadotropin-dependent former medium. In our study, comparable propor(Miller et al., '89). The results of Study I support tions of embryos (2.5-5.5%) reached blastocyst the notion that fertilization rate in vitro is not stage when the culture medium was Ham's F10, affected markedly by the basic constituents of mKRB, or TALP, all of which were supplemented three common culture media. Comparable cleav- with BSA. age rates were achieved despite using media Domestic cat embryos allowed t o culture longer which differed substantially. In contrast, the type than 120 h degenerated, indicating that these emof protein in the co-culture medium significantly bryos experience a block in in vitro embryogeneinfluenced fertilization in vitro. Given that 1)the sis between the morula and blastocyst stage of follicular oocyte has at least 84 h to mature after development. In this context, the domestic cat is a PMSG challenge and 2) complex medium con- similar t o the rabbit, a species in which embryos taining fetal calf serum or homologous serum is also arrest in vitro at the morula stage (Kane and TABLE 2 . Effect ofprotein availability and source on fertilization

~

~~

45/46 a

100 -

39/42 a 29/35 a

U

29/35 a

80 -

5

Eo

I

60-

Morula

W

Blastocyst

P

c3

*

5

40-

U

4

2

20 -

0-

PVA

BSA

FCS

Protein Supplement Fig. 3. Percentage of embryos developing to morulae or blastocysts at 96 h of culture in Ham's F10 containing one of four protein supplements. Numbers above bars indicate total morulaeitwo-cell embryos or total early blastocystsimorulae. a.bxcValueswithin treatments with different superscripts differ (P < .05).

L.A. JOHNSTON ET AL.

356

a

s16 to 32-cells

>8 to 16-cells

4 to 8-cells a

a

a

early blastocysts a

a a

80

60

40

rn

PVA BSA FCS

a s

20

0 24

48

96

72

120

Hours in Culture Fig. 4. Embryo development after 24, 48, 72, 96, and 120 h of culture in Ham's F10 containing one of four protein supplements. a,b,cValueswithin treatments with different superscripts differ (P< ,051.

Foote, '70). Most other species experience a block earlier in development. Bovine and ovine embryos become blocked at eight to 16 cells (Camous et al., '84; Gandolfi and Moor, '87). Mouse embryos (Biggers et al., '62) arrest in vitro at the two-cell stage, hamster embryos (Yanagimachi and Chang, '64; Schini and Bavister, '88) at both the two- and four-cell stage, and pig embryos (Davis and Day, '78) at the four-cell stage. In general, an in vitro developmental block appears related to activation of the embryonic genome (Braude et al., '79; Crosby et al., '88; Frei et al., '89), a transitional period when embryo metabolic requirements are demanding (Biggers, '71; Cross and Brinster, '73). Variations of Krebs-Ringer bicarbonate medium or most media designed for human tissue culture have proved inadequate for culturing hamster, mouse, rabbit, rhesus monkey, sheep, pig, or cow embryos from first cleavage division through the blastocyst stage (Biggers, '71; Tervit et al., '72; Davis and Day, '78; Boatman, '87; Gandolfi and Moor, '87; Kane, '87a,b; Schini and Bavister, '88). In some species, developmental blocks have been overcome by modifying media (Kane, '87b; Chatot et al., '89; Seshagiri and Bavister, '89a,b) or by establishing co-culture systems with oviductal or uterine epithelial cells (Gandolfi and Moor, '87; Eyestone and First, '89b). The ability of domestic cat embryos to overcome developmental arrest and prog-

ress t o the early blastocyst stage appeared partially dependent on protein source. This represents one of the first documented observations suggesting that additional factors, some of which are present in serum, play an essential role in advancing in vitro growth beyond the morula stage. Classical studies by Brinster ('65) demonstrated the importance of pyruvate as an energy source for early-stage mammalian embryos. Sodium pyruvate was included in all of our test media. Lactate, oxaloacetate, and phosphoenol pyruvate also support development of two-cell mouse embryos t o blastocysts (Brinster, '65). However, lactate as the only energy source fails to stimulate development of one-cell mouse embryos to two cells (Biggers et al., '67) and actually is detrimental t o normal first cleavage (Cross and Brinster, '73). Sodium lactate did not appear to be required for in vitro development of cat embryos as 98% of the embryos cultured in Ham's F10 (without Na lactate) advanced t o morulae. For mouse embryos, glucose supports development after the eight-cell stage (Hammond, '49; Brinster, '651, but Chatot et al. ('89) recently demonstrated that the presence of glucose during the first 48 h of culture actually inhibits the number of embryos advancing to blastocysts. Glucose (in the presence of phosphate) also inhibits two-cell and eight-cell hamster embryos from developing into morulae or

CULTURE OF CAT EMBRYOS

blastocysts (Schini and Bavister, '88; Seshagiri and Bavister, '89a). The impact of glucose appears to be species-specific, because the glucose-containing Ham's F10 and mKRB media enhanced cat embryo development compared to TALP (without glucose). However, for cat embryos, the effects of glucose may be subtle and, for this reason, more detailed studies are planned involving the addition and removal of different concentrations of glucose during various embryonic growth phases. In the domestic cat IVF system, the availability and type of protein supplement (Study 11) had a greater impact on fertilization success than medium (Study I). In the absence of protein (the PVA control), fertilization rates were lower, but still more than two-thirds of the ova cleaved. Caro and Trounson ('86) reported successful fertilization and embryo development in a human IVF culture medium without protein. However, in that study as well as our own, it is noteworthy that sperm populations were exposed to minute amounts of protein during swim-up processingkentrifugation procedures. As Car0 and Trounson originally suggested, this brief exposure as well as the presence of follicular fluid proteins in the cumulus oophorus may have been sufficient t o capacitate sperm and promote the fertilization event. In either case, the successful intra-species fusion of human and cat gametes does not appear t o depend on protein availability. Nonetheless, the overall rate of fertilization and subsequent pre-implantation embryo development in the cat was improved by the presence of exogenous proteins. In most conventional embryo culture systems, BSA generally is the macromolecule of choice. The beneficial characteristics of BSA are: 1) the albumin-bound fatty acids that are used as energy substrates; 2) the ability to chelate toxic metallic ions contained in the medium; and 3) the ability to stabilize membranes (Cholewa and Whitten, '70; Kane and Headon, '80; Kane, '87b). It also is possible t o replace either BSA or other types of sera with protein-free macromolecules. Cholewa and Whitten ('70) demonstrated that polyvinylpyrrolidone (PVP)can be substituted for BSA in the short-term culture of two-cell mouse embryos to blastocysts. Bavister ('81) introduced the use of another synthetic polymer, PVA, as a macromolecule for culturing hamster spermatozoa. This synthetic polymer was subsequently used in place of BSA in the culture of hamster embryos from the eight-cell to blastocyst stage (Kane, '87b; Kane and Bavister, '88). PVA also has been demonstrated t o be an effective substi-

357

tute for BSA in the culture of early stage rabbit embryos, although BSA is required for blastocyst formation (Kane, '87b). Traditionally, other types of sera (including FCS, human cord serum, human preovulatory serum) have been used as supplements but their precise role in successful embryo culture remains unclear and controversial at best. Some investigators suggest that serum contains factors which are toxic t o developing embryos (Caro and Trounson, '84; Ogawa and Marrs, '87), whereas others have demonstrated improved embryo development by adding serum (cow, Wright et al., '76; mouse, Saito et al., '84; rhesus monkey, Boatman, '87). In our study, cat embryos were able to develop in vitro in the presence of the non-protein macromolecule PVA. Additionally, cat embryos preferred certain protein supplements to others. Of particular interest was that FCS was superior t o BSA and produced similar preimplantation embryo development t o homologous serum. This suggests that there may be factors in both FCS and ECS which differ from those in BSA and play an important role in enhancing development of cat embryos beyond the morula stage. It appears that in vitro developmental requirements for the two- to 32-cell domestic cat embryo are relatively simple. However, the prerequisites for continued growth are more complex and not yet established. High rates of fertilization (>70%) can be achieved with follicular oocytes and electroejaculated sperm and, although a high proportion of these embryos develop t o morulae, further studies are needed to ensure that these embryos advance t o blastocysts in vitro. Such studies are relevant to understanding the growth and metabolism of carnivore embryos and for the practical use of in vitro fertilized cat oocytes for embryo transfer or cryopreservation.

ACKNOWLEDGMENTS We thank S.L. Hurlbut for her dedicated technical assistance, Dr. E. Jones for veterinary care, and K. James and R. Fry of the National Institutes of Health (NIH) Animal Center for animal care. This project was funded, in part, by grants t o Dr. Wildt from the NIH (NIH 23583) and the Friends of the National Zoo (FONZ). LITERATURE CITED Bavister, B.D., and R. Yanagimachi (1977) The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in uitro. Biol. Reprod., 16:228-237. Bavister, B.D. (1981) Substitution of a synthetic polymer for

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