ELSEVIER

IN VITRO PRODUCTION OF PIG EMBRYOS:

COMPARISONS OF CULTURE MEDIA AND BOARS

C. R. Long, J. R. Dobrinsky and L. A. Johnson

Germplasm and Gamete Physiology Laboratory

Agricultural Research Service, U. S. Department of Agriculture
Beltsville, MD 20705 USA

Received for publication: octoher 1, 1997

Accepted: February 11, 1998

ABSTRACT

The utilization of in vitro produced pig embryos for commercial production or research is
dependent upon the development of improved methodology. Our objective was to establish a
consistent in vitro embryo production (IVP) system and subsequently utilize the procedures to
evaluate culture system components and boar effects, To summarize the IVP system, 403
inseminated oocytes from a total of 2243 were analyzed across 17 replicates for maturation and
fertilization efficiency, while 1838 zygotes were cultured in 26 replicates for developmental
data. Penetration, cleavage and blastocyst development rates were determined at 18,44 and
either 144 or 168 h post insemination, respectively. Monospermic penetration averaged
31.8+7.3% while polyspermy was 30.8fl7.2%. Cleavage rate was 44.9+16.1%, with 21.8*7.5%
of fertilized oocytes and 51.9+15.9% of cleaved embryos developing to blastocysts. For culture
medium comparison, fertilized oocytes were cultured in either BECM-6, BECM-7, NCSU-23 or
NCSU-23aa and supplemented on Day 5 post insemination (pi) with 10% FCS. These treatments
resulted in 4.0,4.9, 19.8 and 13.6% (+ 3.2%) blastocysts by Day 7 pi, with an average cell
number of 44.4f 9.0, 65.1+8.2,61.3&4.5 and 64.4k4.8, respectively. These IVP procedures
consistently produced zygotes from semen of several different boars, capable of forming
blastocysts in vitro. Comparison of developmental rates among the boars indicated that this
system is variable among boars but not strictly boar-dependant. Culture media comparisons
suggest that NCSU-23 yielded a higher percentage of blastocysts than the other media in this IVP
system.
PuMished by Elsevier Science Inc.
Key words: pig, oocyte, in vitro, fertilization, culture

Acknowledgments
We thank B.N. Day, H. Funahashi, L. Abeydeera and M. Yoshida for assistance in
establishing the IVP system; K. Wells for assistance in culture media modifications; and L
Schreier for expert technical assistance.

Theriogenology 51:1376-1390, 1999 0093-691X/99/$-see front matter

Published by Elsevier Science Inc. PII SOO93-691X(99)00081-3
1376 Theriogenology

INTRODUCTION

The availability of in vitro production (IW) techniques for bovine embryos has advanced
the understanding of early embryonic processes, permitted the use of large numbers of
affordable embryos to be utilized in subsequent experiments and contributed to the
commercialization of bovine embryo production. These techniques have not been developed to
suitable levels in swine. The increased interest in using swine as a model system in
biotechnology applications and research efforts such as sperm sexing, necessitates increased
focus on improving in vitro embryo production systems. A practical IVP system requires the
collection of large numbers of developmentally competent oocytes that undergo cumulus
expansion and nuclear and cytoplasmic maturation. Upon insemination oocytes should exhibit
consistently high sperm penetration with low polyspermic fertilization. Furthermore, a
competent embryo culture system is necessary to develop desirable embryos for selection or
transfer. In practice these objectives have been difficult to achieve (26,27,39).

In vitro production of pig embryos has been plagued by a high incidence of polyspermy,
failed or delayed paternal pronucleus formation and decreased development upon culture
(8,29,4345). Improvements in maturation ( 11,17,18,3 1,47) and fertilization ( 13,25,35) systems
have overcome some previous limitations and led to high nuclear maturation rates and better
male pronucleus formation. However, polyspermy is still a prevalent obstacle in most IVP
systems. Multiple methods have been employed to reduce polyspennic penetrations, however,
few have been successful in maintaining high sperm penetration combined with low polyspermy
(4,6,15,28). In vivo and in vitro, cortical granules are well distributed and released from oocytes
following sperm penetration (7,2 1,42), inducing the block to polyspermy. The prevalence of
polyspermy in vitro may reflect the inability of porcine oocytes to develop a quick, substantial
block to supemumerous spermatozoa (19,30,34). Since polyspermy is deleterious in mammalian
fertilization, these abnormally penetrated oocytes lead to decreased development rates of
cultured, in vitro produced embryos (13,45).

Culture of in vivo produced 1 to 2 cell pig embryos has been extensively studied and
thoroughly reviewed (32,33,37). Although standardized media are available for the successful
culture of embryos to morula and blastocyst stages, the cell numbers of resulting embryos are
low (10,32). Furthermore, transfer of embryos cultured in vitro to recipient gilts results in low
pregnancy rates and increased embryonic mortality (12).

The objective of these experiments was to develop techniques for the production of large
numbers of embryos in vitro and utilization of those embryos for testing culture media and
culture media modifications. The embryo production system was then used to evaluate
development rates using sperm from several boars. Establishing the IVP system is consistent
with laboratory objectives of providing a cost effective source of pig embryos for related
research goals and of producing sex selected embryos following gender selection of
spermatozoa (36).
Theriogenology 1377

MATERIALS AND METHODS

In Vitro Maturation

Ovaries were collected at a local abattoir from prepuberal gilts, washed in 0.9% saline
and transported to the laboratory in saline within 6 h post mortem at 25°C. Upon arrival, the
ovaries were washed with room temperature saline and held in same until aspiration. Cumulus
oocyte complexes (COC) were aspirated from 2- to 8- mm follicles using an 18-g short bevel
hypodermic needle attached via a glass cannula to a 50-mL polystyrene conical tube (Falcon).
This tube was in line to a vacuum pump set to a flow rate of approximately 45 mL of Hz0 per
minute. Three to six technicians aspirated follicles simultaneously for up to 1.5 h. Cumulus
oocyte complexes were allowed to settle for approximately 7 min at room temperature and
follicular fluid was removed. All COC were washed twice with Hepes-PVA (2; Table 1)
allowing the COC to settle each time. Following the final wash, COC with multiple layers of
intact cumulus were selected for maturation at 25°C washed twice more in Hepes-PVA and
washed 3 times in maturation medium prior to culture. Maturation medium (MAT) was based
on BSA-free NCSU-37 (33) supplemented with 5 mg/mL insulin (mNCSU-37) and used within
10 d of preparation. On the day of use, maturation medium was prepared by adding porcine
follicular fluid to 10.0% (v/v; 14) 50 ,uM b-mercaptoethanol, 0.6 mM L-cysteine, and IO @ml
epidermal growth factor (EGF) to the base medium. The maturation medium was filter sterilized
and equilibrated for at least 2 h prior to use. Fifty to sixty COC were cultured per well in 500 PL
of maturation medium in 4 well tissue culture plates (Nunc, Roskilde, Denmark). Immediately
after moving oocytes to maturation medium wells, 10 RJ/mL eCG, IO IU/mL hCG (Intervet
America Inc., Mill&no, DE), and 1.0 mM db-CAMP (Sigma, St. Louis, MO) were added from
stock solutions (1000 IU/mL hCG and eCG, 100 mM db-CAMP all in H20). After placing CGC
to maturation, all oocyte handling outside the incubator was performed on a heated microscope
stage at 38°C. The oocytes were then cultured for 22 h at 38.7”C in a humidified atmosphere of
5.0% CO2 and air. Oocytes with expanded cumulus complexes were washed and then cultured
an additional 22 h in fresh maturation medium without eCG, hCG and db-CAMP prior to
fertilization.

Sperm Preparation

Semen was collected from mature, fertility tested boars once or twice per week by the
gloved hand method. Semen was collected as the sperm rich fraction and immediately
transported to the laboratory. To avoid reported boar effects (40,41), only 1 boar was used for
the culture media comparison study. Semen was transferred to a 15-mL polystyrene centrifuge
tube (Falcon) and 10 pl/mL of antibiotic/antimitotic solution (Gibco) was added and then placed
in a room temperature water bath. The semen was allowed to slowly cool by placing the water
bath at 18°C in storage until use. Semen was generally held 4 to 5 h, but could be used
immediately following collection. One ml semen was diluted in 9 mL 0.9% (v/v) saline
containing 100 rnfl BSA (Sigma # A-7906), and 100 mg/L kanamycin sulfate (saline-BSA)
then washed by centriti@ion in a 15 mL polystyrene conical tube (Falcon) at 900 x g for 5 min
1378 Theriogenology

at room temperature. The saline-BSA was removed and the sperm pellet resuspended in 10 mL
of saline-BSA, and this procedure was repeated twice. Washed spermatozoa were resuspended
in 3 mL of modified TLP-PVA (2; Table 1) with 3 mg/mL fatty acid-free BSA (A-6003;
mTLP-PVA) and held in a 4-mL polystyrene tissue culture tube at 38.7”C in 5.0% CO2 and air
for about 15 min until use. The concentration of resuspended spermatozoa was determined by
hemocytometer, and motility was assessed by visual observation, using a heated microscope
stage, by a single technician.

In Vitro Fertilization

Fertilization was performed in mTLP-PVA with 2 mM caffeine sodium benzoate (FERT

[46] and M. Yoshida, personal communication). Medium drops were prepared by placing three 2
to 3 PL base drops in a 35-mm Petri dish (Falcon), covering with 3-4 ml light mineral oil
(Sigma), and adding 90 PL of additional medium to each starter drop. Fertilization drops were
equilibrated at least 2 h prior to adding oocytes and spermatozoa.

Oocytes cultured for a total of 44 h in maturation medium were stripped of cumulus by

addition of 500 FL of 0.15% hyaluronidase in equilibrated mNCSU-37 to each well and gentle
aspiration with a small bore pipette. Oocytes were washed 3 times in FERT and moved to
fertilization drops. The oocytes were then added in 2 to 3 PL of medium and held in fertilization
drops until addition of spermatozoa.

Spermatozoa in mTLP-PVA were mixed by inversion and gentle vortexing, and were
then diluted to 4 x 10’ motile sperm cells in 2 mL FERT in a polystyrene tissue culture tube.
Five microliters of spermatozoa diluted in FERT were introduced to fertilization drops
containing 50 oocytes each. The final sperm concentration was 2 x lo4 motile sperm/ml,
corresponding to approximately 40 motile sperm cells per oocyte. Spermatozoa and oocytes
were coincubated at 38.7”C for 6 h in humidified atmosphere of 5.0% CO* and air.

Culture Medium and Conditions

Presumptive zygotes were removed from FERT and washed twice in the respective
embryo culture medium using a small bore pipette. Embryos were cultured in groups of 30 to 50
in 4-well plates containing 500 PL of medium per well.

Chemically defined, protein-free Beltsville Embryo Culture Medium (BECM-3; lo), was
supplemented with 4 mg/mL heat shock fraction V BSA (Sigma # A-7906) and was designated
BECM-6. The BECM-6 was supplemented with 7.0 mM taurine and 5.0 mM hypotaurine to
produce BECM-7. The NCSU-23 was prepared according to the published formulation of
Petters and Wells (33) using heat shock fraction V BSA (Sigma A-7906). The NCSU-23aa was
modified from NCSU-23 by adding MEM and BME amino acids (Sigma) at 10.0 and 20 ml/L,
respectively. Culture media components are listed in Table 2 for comparison purposes. At 125 h
post insemination, all embryos were washed and transferred to fresh wells of the respective
culture medium, with 10% heat-inactivated FCS (HyClone) replacing BSA as the protein source,
Theriogenology 1379

Table 1. Chemical composition of media used during in vitro maturation and fertilization of
porcine oocytes.

Ingredient (mM) Hepes-PVA* mmP-PVA* NCSU-37**

NaCl 114.00 114.00 108.73

KC1 3.20 3.16 4.78

CaC12’2HzO 2.00 4.7 1.70

lQw34 0.34 1.19

MgS04 7H20 1.19

NaH2PO;H20 0.35

MgC12’6Hz0 0.50 0.50

NaHCO, 2.00 25.00 25.07

HEPES 10.00

GlllCOSZ 5.00 5.55

Sodium lactate 10.00 10.00

Sodium pymvate 0.20 0.01

Glutamine 1.00

Sorbitol 12.00 12.00

BSA (mg/ml) 3.00 4.00

PVA (mg/ml) 0.10 1 .oo

Geutamycin (ml/L) 0.5

Penicillin G (mg/ml) 65.0

Amikacin Sulphate 1.0

Phenol Red (g/L) ,001

*Modified from Bavister (2)
**NCSU-37 as published by Petters and Wells (33)
1380 Theriogenology

Table 2. Chemical composition of media used during in vitro culture of porcine embryos.

Ingredient (UIM) BECM-6’ BECM-$ NCSU-23b NCSU-23’

NaCl 94.59 94.59 108.73 108.73

KC1 6.00 6.00 4.78 4.78

CaQ2H20 1.71 1.71 1.70 1.70

m2po4 1.19 1.19

MgS04 7HZ0 1.19 1.19 1.19 1.19

NaHCO? 25.07 25.07 25.07 25.07

Glucose 5.56 5.56 5.55 5.55

Sodium lactate 23.00 23.00

Sodium pyruvate 0.33 0.33

Glutamine 1.00 1.00 1.00 1.00

Taurine 7.00 7.00 7.00

Hypotaurine 5.00 5.00 5.00

MEM amino acids (ml/L) 10.00 10.00 10.00

BME amino acids (ml/L) 20.00 20.00 20.00

BSA (mg/ml) 4.00 4.00 4.00 4.00

Phenol Red (@L) .OOl ,001

a BECM-6 and -7 based on BECM-3 (Dobrinsky; 10).
b NCSU-23, as published by Petters and Wells (33).

and cultured to 168 h post insemination, For boar comparisons, embryos were produced as
described and cultured in NCSU-23 without modification. No FCS was added at day 5 pi and
embryos were evaluated for blastocyst development on day 6 pi.
Theriogenology 1381

MorphologicalAssessment
of Embryos

A subsetof fertilized oocyteswasrandomlyremovedfrom culturemediumat 18h post

insemination to evaluatespermpenetrationandpronucleardevelopment.Theseovawerefixed
in 2%paraformaldehyde in PBSwith 10%Triton X-100 for I h at 37°C. After washingin PBS,
oocyteswereplacedona glassslidein 10,uLPBSwith 20%glycerol,2 &mL Hoechst33342
and100mg/mLDABCO,pH 9.0. A glasscoverslipwasplacedover thedropandaffixed with
clearnail polish.The zygoteswereevaluatedona ZeissAxiovert-I 35 microscope usinga
Hoffman-Zeiss objective(x 40). Epifluorescence
illuminationfrom a 100W mercurybulbwas
usedto excitethefluorochromewith a Zeissfilter setspecificfor UV (#487901;3651).
Oocytesdisplayingmeioticstructuresprior to metaphase II or fertilizedovawith abnormal
chromatinstructures wereconsidered incompetentfor furtherdevelopment.Pronucleiwere
countedto determinemonospermy andpolyspermyrates.

Cleavagerateswereevaluatedat 44 h by countingthenumberof inseminatedoocytes

that haddividedevenlyto 2- to 8-cellembryos.Depending on theexperimentalendpoint,
embryoswereevaluatedat 144h or 168h postinsemination.Cavitatedembryosthat exhibited
distinctinnercell massandtrophectodermcell layerswereconsideredasblastocysts
and
expandedblastocysts.All embryoevaluationswereperformedona heatedmicroscope stageat
38°C.

Forculturemediacomparisons, blastocystsandhatchedbiastocystswereevaluatedfor
total cell number.Followingembryoassessment at 168h, embryoswerefixed in 2.0%
paraformaldehyde in PBS(pH 7.0) for 2 h at 37°C. After washingin PBS,embryoswereplaced
in mountingmediumasdescribed above.Fluorescent imageswerecapturedon screenusinga
videocamera(ZeissZVS-UDEC). Thefreeze-framed imageon thevideomonitorwasusedfor
cell counts,whichwasperformedby a singletechnician(10).

StatisticalAnalysis

DatawereanalyzedusingSAS(v. 6.12;SASInstituteInc., Cary,NC). Culturemedia

comparisons weremadeby calculatingcleavageanddevelopment ratesfor eachreplicateand
thepercentage dataanalyzedusingthe mixedmodel.Culturemediumwasconsidered the main
effect. Replicatewasusedasa randomvariablewith compoundsymmetrycovariancestructure.
Leastsquares meansandstandarderrorsfor cell numbers
werecalculatedfrom individual
embryosusingculturemediumasthemaineffect andreplicateasa randomcomponent by the
mixedprocedure.Main effect for eachvariablewastestedusinganF-test, Leastsquaresmeans
andstandarderrorswerecalculatedfor presentation,andcomparisonbetweengroupswasdone
by Student’st-test.

Summarystatisticsfor the in vitro embryoproductionsystemwerecalculatedfrom a

total of 16replicates,with 26 observations for cleavageanddevelopment
dataandfrom I1
concurrentreplicateswith 17observations for maturationandspermpenetrationdata. Datafor
boarcomparisons aresummarized from2 to 4 replicatespersire.
1382 Theriogenology

RESULTS

In Vitro Maturation, Fertilization and Culture

Oocyte penetration and development data, collected from control groups in 11 replicates,
are shown in Table 3. Meiotic maturation of oocytes was high (87.6%), as evidenced by
metaphase II chromatin or 2 polar bodies and at least 2 pronuclei. Single or multiple sperm
penetrations occurred equally in penetrated zygotes under these fertilization conditions.
Condensed sperm chromatin in the cytoplasm of penetrated oocytcs was rare (data not shown),
indicating paternal pronuclei formed at a high rate. At 44 h post insemination, 44.9% of
fertilized oocytes cultured had cleaved evenly to at least the 2-cell stage. Of 1838 presumptive
zygotes placed into NCSU-23,372 (2 1.8%) developed to the blastocyst stage by 144 h post
insemination. Blastocyst development calculated from cleaved embryos was 5 1.9%.

Table 3 also shows the cleavage and blastocyst rates as calculated from penetrated
oocytes. According to the sperm penetration data, 13 12 of the 1838 zygotes were predicted to be
penetrated by at least 1 spermatozoa and 584 were estimated to be monospermically fertilized.
The cleavage rate of zygotes was higher than the predicted rate of monospermy. Cleavage and
blastocyst development rates were 76.5 and 28.4%, respectively, when calculated from the total
number of penetrated oocytes.

Culture Media Comparisons

The above data indicate that mature pig oocytes can be produced in sufficient numbers
with acceptable developmental competence to be useful for further experimentation. The data in
Table 4 compares pig embryo culture medium BECM-6 and NCSU-23 with modified versions of
these media.

Cleavage rates in the tested media were not different (P = 0.53). However, development
rates to blastocyst at 168 h were statistically different (P < 0.01). Embryos cultured in NCSU-23
tended to show a better rate of development to the blastocyst stage than NCSU-23aa (P = 0.06),
but both these media outperformed BECM-6 and BECM-7. Adjusting the blastocyst rate to
account for the small differences in the cleavage rate only enhanced the differences between
groups. Culture in NCSU-23 was superior to that of all other groups (P < O.Ol), and NCSU-23aa
promoted blastocyst development at a higher rate than BECM-6 or BECM-7 (Table 4). Culture
medium had no significant effect on cell numbers of blastocysts at 168 h post insemination (P =
0.20). However, BECM-6 tended to produce blastocysts with lower cell numbers the other
media. Least squares mean for BECM-6 was more than 16 cells per embryo lower than in other
culture treatments, and the lack of a statistical difference may be due to the low number of
embryos (n = 13) produced in BECM-6.

Boar Comparisons

In addition to the standard boar for IVF, 6 additional boars from the breeding herd were
also utilized for insemination of IVM oocytes. At least 2 replicates were performed for each
Table 3. In vitro maturation, penetration and development rates for the pig embryo IVP system.

Inseminated oocytes

Total No. of % of % of % No. of % of % of % of

No. of Fixed Nuclear Monospermic Polyspermic Cultured cleaved Blastocysts Blastocysts/
oocytes oocytes maturation oocytes oocytes (Day 6) Cleaved oocytes
inseminated

2243 405 87.9 31.8 30.8 1838 44.9 21.8 51.9

+ 6.7 * 7.3 + 17.2 +. 16.1 k7.5 f 15.9

Oocytes predicted to be penetrated

Predicted to be % of % of % of
penetrated cleaved oocytes Blastocysts (Day 6) Blastocysts/monospermic

1312 76.5 28.4 70.6

Summary statistics only.
1384 Theriogenology

Table 4. Developmental rates of in vitro produced porcine embryos: comparison of different

culture media.

Culture Number of Percent Percentage of Percentage of Mean cell

medium cultured cleaved blastocysts, blastocysts number of
ooc yte? Day 7 @I from cleaved Day 7
-ytes blastocysts
5 SEM
BECM-6 328 44.8 4.0a (13) 8.7a 44.4 + 9.0
BECM-7 342 45.6 4.9a (17) 9.Y 65.1 + 8.2
NCSU-23 345 39.8 19.gb (68) 49.0b 61.3 54.5
NCSU-23aa 422 45.4 13.6b (57) 29.3’ 64.4 2 4.8
+ SEM + 8.6 + 3.2 +- 3.7
&Total of 3 replicates.
Least Squares Means are shown + Standard Error of the Mean (SEM)
Values within a column with different superscripts are statistically different, P < 0.05.

sire, and the data are summarized in Table 5. All boars were capable of penetrating oocytes
under the described conditions, and the penetrated oocytes were capable of initiating cleavage
and development to the blastocyst stage across all sires.

Table 5. Comparison fertilization and development rates of different boars within the in vitro
production system.

Boar No. of % of % of No. of % of % of % of

fixed embryos embryos cultured cleaved blastocysts, blastocysts/
embryos with with oocytes oocytes Day 6 cleaved
2PN PPN oocvtes
A 29 13.8 3.4 248 20.73 4.44 42.13
B 29 31.0 55.2 285 48.31 24.21 59.90
C 74 29.7 37.8 200 40.35 17.50 42.00
D 80 22.5 36.3 202 41.19 15.35 43.73
E 49 22.4 61.2 174 49.14 16.67 35.12
F 46 23.9 13.0 156 28.61 10.02 29.76
Summary statistics only.
Theriogenology 1385

DISCUSSION

In this study, we made modifications to the published procedures for in vitro production
of porcine embryos and then evaluated both culture media and boar selection as components of
the system. The data presented show that pig embryos can be produced in vitro and established
that culture medium can support development to the blastocyst stage. However, as expected,
boar variation was high, and polyspermy remains a significant problem. Moreover, IVP
blastocysts were reduced in overall cell number when compared with similar stages in vivo,
likely due to insufficient embryo culture conditions.

The in vitro maturation system was established using modified protocols of Funahashi
(11,12,16,17). High rates of nuclear maturation were observed, and sperm-penetrated oocytes
generally showed high rates of male pronucleus formation, indicating sufficient male pronucleus
growth factor (14,22). This is similar to findings in previous reports that in NCSU-23 based
medium, addition of cysteine and P-mercaptoethanol raises intercellular glutathione
concentrations, leading to increased pronuclear formation following fertilization ( I7,18).
Addition of db-CAMP and EGF has also been described as having a beneficial etrect on oocyte
maturation, probably via cumulus cell interactions (5,9,3 I).

The in vitro fertilization system was based on protocols of Yoshida (46 and personal
communication). The mTLP-PVA based system produced lower rates of polyspermy compared
with that of traditional TCM-199 based media in early trials, and it utilized a lower sperm/oocyte
ratio during coincubation. In the present experiments, we utilized freshly collected semen
without the need for overnight storage, as reported in previous studies (2 1,25,29). Fertilization
medium utilized fatty acid-free BSA, and there was no significant preincubation of spermatozoa
in the fertilization medium or in sperm pre-incubation medium prior to insemination (4,15).
Coincubation of spermatozoa and oocytes was 6 h based on previous reports (6). Overall, the
sperm penetration rate of 62.6% was lower than that reported by other investigators ( 12,26), but
did not require co-culture with oviductal cells or oviductal fluid (2 I ,29).

Calculation of monospermy and polyspenny rates from the total number of oocytes
penetrated shows that the ratio is nearly 1: 1, From the 62.6% penetration rate, approximately
I3 12 of the cultured oocytes were predicted to be penetrated by at least one spermatozoa. The
cleavage rate estimated from the oocytes predicted to be penetrated results in a value higher than
the monospermic penetration rate. This would suggest that polyspermic and perhaps a low
proportion of parthenogenetic oocytes also cleaved to early stages. It is not known if these
polyspermically fertilized oocytes developed to blastocysts.

Boar and ejaculate variation has been a problem in maintaining high penetration and
development rates in vitro. Analogous to previous reports, boars in this study did vary
considerably, indicating that all sires did not respond to in vitro conditions equally (40). No
attempt was made in our study to adjust fertilization conditions in order to increase fertilization
and development rates for sires. We conclude that the system described is tolerant to boar and
ejaculate variation but adjustments in sperm numbers, caffeine concentration and sperm oocyte
1386 Theriogenology

incubation time should be determined for each sire in order to optimize a boar’s potential for in
vitro fertilization,

The culture media used in this study were distinguished by minor modification of the
components. Other than slightly higher Na+ and elimination of PO4-, BECM-based media have
essentially the same salt formulation as NCSU-23. The differences between media were in the
presence or absence of lactate, pyruvate, taurine, hypotaurine or essential and nonessential
amino acids. The BECM-6 medium differs from NCSU-23aa by the addition of lactate and
pyruvate, and the omission of taurine and hypotaurine. These changes resulted in a significant
decrease in blastocyst development, and tended to display lower cell numbers per embryo.
Adding taurine and hypotaurine to BECM-6 (BECM-7) did not improve development to the
blastocyst stage; however, the ceil numbers of resulting embryos was similar to those of the
NCSU-23 based media. Addition of amino acids to NCSU-23 (NCSU-23aa) at the same rate as
BECM media resulted in decreased development of cleaved embryos but had no effect on cell
number.

The source of BSA is important to the successful production of embryos in vitro. In the
previous study (10) comparing NCSU-23 and BECM-3, the BSA was fraction V prepared by
cold ethanol precipitation. In the present study, BSA prepared by heat shock was used in the
same concentration across all culture treatments. The lack of effect by addition of amino acid to
NCSU-23 may be due to the presence of BSA or to detrimental amino acids of the full
supplementation (3). In a completely defined medium, the effects of full amino acid
supplementation would likely be different.

In the context of this study, full supplementation with essential and nonessential amino
acids seemed to exert a negative effect on embryo development. This supports previous work on
hamster embryos which indicated that certain amino acids can be toxic (3,38). However, with in
vivo produced zygotes and 2-cell embryos, full amino acid supplementation was not detrimental
to embryonic development in either defined or BSA supplemented BECM-based pig embryo
culture medium (IO), suggesting that amino acid requirements may differ for IVP embryos or
the in vivo produced embryos are more tolerant to adverse culture conditions. Alternatively, the
medium components of BECM may actually be detrimental to early IVP zygote development.
Further research is needed in porcine maturation, fertilization and embryo culture to define the
amino acids that may have beneficial or detrimental effects on embryo development.

Adding the amino acids taurine and hypotaurine to BECM-6 tended to increase blastocyst
cell number. Addition of taurine in modified KSOM bovine culture systems has been shown to
be beneficial, and is likely related to oxygen tension during culture (23,24). Similarly, the
increased in vitro development observed in our present study is similar to previous results (1,32)
that demonstrated a significant improvement in pig and hamster embryo development in the
presence of taurine and/or hypotaurine.

A comparison of the culture media in this study does not agree with previously published
data from our laboratory comparing BECM3 to NCSU-23 during the culture of in viva-derived
embryos ( 10). Dobrinsky et al. ( 10) cultured in vivo-derived embryos from Day 2 to Day 8
Theriogenology 1387

using either BECM3 or NCSU-23 and reported higher rates of development in BECM-3. In
contrast, IVP embryos in this study failed to develop successfully in BECM-6 and showed the
best development in NCSU-23. Preliminary experiments with IVP embryos indicated no
difference in development rates when cultured in BECM-3 or BECM-6 (data not shown).
Holding zygotes in mTLP-PVA for an additional 24 h after insemination is inhibitory to
development, whereas moving them from NCSU-23 to BECM-6 after the morula stage maintains
development and results in good quality blastocysts (data not shown). This suggests that the
effects of BECM-6 on porcine embryos could be stage-specific. Petters and Wells (33) have
reviewed the role of lactate and pyruvate in embryo culture media, and have suggested that
lactate and pyruvate appear to be detrimental to porcine embryo development in the presence of
glucose (33). In total, this would indicate the lactate and pyruvate components of BECM-6 may
be detrimental in the presence of glucose, and this is manifested in zygotic or early cleavage
stages of IVP embryos, However, no experiments were performed using NCSU-23 with added
lactate and pyruvate to test this hypothesis.

One objective of our work is to develop methods for producing gender preselected
offspring. In previous work we demonstrated the effectiveness of flow cytometry sperm sorting
for separating X- and Y-bearing spermatozoa (20). We have also used the procedures described
in this paper to complement our sperm sorting protocols (USDA-Beltsville Sperm Sexing
Technology) for the production of numerous litters of sex-selected piglets (unpublished data)
from embryos produced using sorted boar spermatozoa under the conditions described in this
study.

In summary, pig oocytes can be successfully matured and fertilized in vitro using these
procedures, resulting in adequate levels of monospermic penetration. Zygotes can be cultured
successfully in a biphasic system using NCSU-23 medium in early development with
substitution of FCS for BSA following the morula-blastocyst transition at 125 h post
insemination. Further moditications of the system are necessary to eliminate boar and ejaculate
variation, as well as to further decrease polyspermy. However, the results of this study are
sufficient for successful in vitro production, using several different boars, of embryos and
zygotes for further experimentation.

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