thousands of calves have been produced, problems with abortions, increased birth Ž
weights, dystocias and higher rates of neonatal mortality have been widely reported for .
reviews, see Walker et al., 1996; Farin and Farin, 1997; Kruip and den Daas, 1997 . Ž
. Walker et al. 1992 first described increased birth weights, lengthened gestation periods
and higher neonatal mortality in sheep pregnancies resulting from in-vitro-derived pregnancies. Subsequently, the inclusion of serum during the in vitro culture of ovine
Ž embryos was implicated in these abnormalities Thompson et al., 1995; Holm et al.,
. 1996 . As a result, there have been a number of recent studies directed at more clearly
Ž . defining the problem s of the inclusion of serum in the in vitro culture of both ovine
Ž and bovine embryos Popovic et al., 1997; Thompson et al., 1998; Blondin et al., 1999;
Jacobsen et al., 1999; Sinclair et al., 1999; Tricoire et al., 1999; van Wagtendonk-de .
Leeuw et al., 1999 . It remains to be demonstrated that any of the currently used in vitro systems result in the production of a statistically significant improvement in the number
of ‘normal’ pregnancies. The present study was organized to determine the efficacy of the in vitro culture of bovine zygotes with and without somatic cell coculture and with
or without serum for the first 72 h of culture. In addition, a survey was conducted on whether the restriction of serum during the first 72 h of development in Menezo’s B2
´ ´
Ž .
Ž .
B2 –buffalo rat cell BRL coculture improved the normalcy of pregnancies resulting from the transfer of embryos.
2. Materials and methods
2.1. Oocyte recoÕery and in Õitro maturation IVM For Experiments 1 and 2, bovine ovaries were transported from a slaughterhouse in a
plastic bag, with no additional fluid added, within an insulated box at a temperature of 26–308C. Upon arrival in the laboratory, the ovaries were rinsed with 288C tap water
Ž and placed for 5 min into 288C tap water containing 1 Nolvasan solution Aveco, Fort
. Ž
. Dodge, IA and 1 7 = cleaning solution ICN Biochemicals, Costa Mesa, CA , after
which the ovaries were rinsed copiously with tap water and maintained at ambient Ž
. temperature 23–278C prior to aspiration. A 19-gauge 3r4-in. needle attached to a
Ž .
Pioneer Pro-Pump Pioneer Medical, Madison, CT was used to aspirate the contents of all follicles between 2 and 10 mm in diameter into a 50-ml centrifuge tube.
For Experiment 3, oocytes were aspirated from cows, primarily Holsteins, with a 5-MHz sector scanning transducer housed together in a vaginal probe with a stainless
steel guide containing a 17-gauge single lumen needle. Aspiration pressure was provided by a foot-pedal-operated Pioneer Pro-Pump set at approximately 50 mm Hg. This
experiment actually represents, in part, a retrospective analysis of the in vitro embryo production and transfer program provided for the owners of donor cattle housed at Em
Ž Tran. The follicular aspirate was rinsed with PBS through an Em-Con filter Immuno
. Systems, Spring Valley, WI . For Experiments 1 and 2, all oocytes with three or more
compact layers of cumulus cells were utilized for maturation and were not graded on the basis of cytoplasmic appearance, whereas in Experiment 3, all OPU oocytes with one or
more layers of cumulus were utilized. All culture procedures, including IVM, fertiliza-
Ž tion and culture were conducted in four-well plates with no oil overlay Nunclon,
. Roskilde, Denmark . Unless stated otherwise, all chemicals were obtained from Sigma
Ž .
St. Louis, MO . Following aspiration of slaughterhouse ovaries or OPU on donor cows, oocytes were
Ž rinsed five times in a modified Tyrode’s Medium
TAL-HEPES; Bio Whittaker, .
Walkersville, MD . Oocytes were placed into maturation medium within 5–9 h after the ovaries were collected at the slaughterhouse or within 30 min after OP. Groups of 30–35
experimental oocytes, or all the oocytes from individual donors were matured for 21–24 h in 0.5 ml TCM 199 with Earle’s salts, supplemented with 2.2 grl sodium bicarbonate
Ž
. Ž
Gibco BRL, Grand Island, NY , 10 heat-inactivated fetal calf serum FCS; Hyclone .
Ž Laboratories, Logan, UT , 4 mg FSH and 6 mg LH Sioux Biochemicals, Sioux Center,
. IA . The oocytes were incubated at 398C in at humidified atmosphere of 5 CO in air.
2
2.2. In Õitro fertilization IVF At the end of IVM, oocytes were rinsed twice in TAL-HEPES and placed in 0.5 ml
of fertilization medium, which consisted of modified tyrode–lactate–pyruvate medium Ž
. TALP; Bavister and Yanagimachi, 1977 . Frozen semen was thawed in a 358C water
Ž .
bath and layered on a discontinuous gradient of Percoll Sigma in a 15-ml centrifuge tube. The Percoll gradient was composed of 2 ml of 90 Percoll overlaid with 2 ml of
Ž 24 Percoll, both of which were prepared with tyrode–lactate TL-HEPES; Parrish et
. al., 1988 . After 30 min of centrifugation at 700 = g, the sperm pellet was recovered,
resuspended in SP-TALP and the sperm concentration determined with a hemocytome- ter. Depending on the bull, sperm were added to fertilization wells at a concentration
ranging from 0.1 to 0.5 = 10
6
rml. At the time, spermatozoa were added to the fertilization wells, 20 mM penicillamine, 10 mM hypotaurine and 1 mM epinephrine and
2 mgrml heparin were also added. After 18 h in IVF, oocytes were removed, rinsed twice in TALP, vortexed for 2 min to remove cumulus cells and then placed into in vitro
culture.
2.3. IVC 2.3.1. Experiment 1
Following vortexing, oocytes aspirated from slaughterhouse-derived ovaries were Ž .
Ž placed in 0.5 ml of one of three media: 1 B2 medium Laboratoire C.C.D., Paris,
. Ž . Ž .
France ; 2 B2 containing 10 FCS; or 3 a coculture system consisting of B2 Ž
. Ž
containing 10 FCS Hyclone on a monolayer of Buffalo Rat Liver cells American .
Type Culture Collection, Rockville, MD . The BRLs were plated at a concentration of approximately 200,000, 100,000 or 50,000 cells at 24, 48 or 72 h before use. On the
fourth day of IVC, the embryos were transferred to fresh media or coculture wells for all three treatments. Two replicates were conducted.
2.3.2. Experiment 2 Oocytes derived from slaughterhouse-derived ovaries were cultured in B2–BRL
Ž .
coculture either with or without 10 serum during Days 1–4 72 h of IVC. After Day
4, all oocytes were transferred to fresh coculture wells containing 10 serum. Six replicates were conducted.
2.3.3. Experiment 3 Oocytes from the donor cows were allocated randomly to B2–BRL coculture either
with or without 10 serum. On Day 4 of IVC, all embryos were transferred to B2–BRL coculture including 10 serum. During the time period over which Experiment 3 was
Ž .
conducted 15 months , all embryos from an additional group of Holstein cows were Ž
. cultured exclusively on TCM 199–BRL coculture with 10 grl BSA A-4503, Sigma
and 10 FCS. In both cases, incubation was conducted at a temperature of 398C in a humidified atmosphere of 4 C0 in air. Most of the resulting embryos were transferred
2
into recipients as described below. 2.4. EÕaluation of embryos
Ž Embryos were evaluated for stage M s morula, EB s early blastocyst, MB s mid-
. Ž
blastocyst, XB s expanded blastocyst, HB s hatched blastocyst and grade excellent– .
Ž .
good s 1, fair s 2 as described previously Hasler et al., 1995 . 2.5. Staining of embryos
All blastocysts were classified for stage and grade and fixed onto slides with Ž
. methanol–acetic acid King et al., 1979 . Following fixation, embryos were stained for 8
min in 10 giemsa, washed three times in distilled water, air-dried and coverslipped. Interphase, mitotic and pyknotic cell counts were determined by an evaluator who was
Ž .
unaware of treatment, grade or stage assignments Farin et al., 1997 . 2.6. Transfer of embryos
Embryos were removed from culture medium on Day 7 and rinsed in TALP prior to loading in a 1r4-ml straw. Single embryos were transferred nonsurgically to the uterine
horn ipsilateral to the corpus luteum of Holstein heifers that had been observed in estrus Ž
. Ž
. between 2 days before q2 and the same day as the IVF of the embryos 0 synchrony .
Pregnancy was determined by rectal palpation at approximately 55–60 days of gestation. The sex of many of the pregnancies produced in this study was determined by
ultrasound examination at Em Tran before the recipients were transported to the farms of the donor owners. Also, whenever possible, the sex of the resulting calves was
recorded. In addition, prior to transfer, biopsies were removed from 296 embryos via
Ž .
micromanipulation and sex was determined by PCR Bredbacka et al., 1995 . 2.7. Data analysis
Differences between and within treatments in embryo production, sex ratio, preg- nancy rate, abortions and the number of calves dead at birth were determined by
Table 1 Effect of B2–BRL coculture vs. cell-free B2 with or without serum on blastocyst production and the
proportions of different stages of blastocysts on Day 8 of culture Media
B2 B2qserum
B2–BRL coculture
a b
c
Ž . Ž
. Ž
. Ž
. No. blastsroocytes
17r303 5.6 13r73 17.8
79r168 47.0
a b
b
Ž .
Ž .
Ž .
Ž .
No. EB of total blasts 9 52.9
2 15.4 4 5.1
a a
b
Ž .
Ž .
Ž .
No. MB 6 35.3
4 30.8 8 10.1
a b
b
Ž .
Ž .
Ž .
No. XB 2 11.8
7 53.8 32 40.5
Ž .
No. HB 35 44.3
a,b,c
Means in rows without common superscripts differ, P -0.05.
chi-square analysis. Differences between treatment in stage, grade the number of total cells and pyknotic cells per embryo was determined by ANOVA and separate means
were then analyzed by Tukey’s HSD test.
3. Results
