R.L. Monson

Degeneration of cryopreserved bovine oocytes via apoptosis during subsequent culture.

Cryopreservation causes a significant proportion of bovine oocytes to undergo degeneration during subsequent culture. We investigated the degeneration mechanism of cryopreserved oocytes. In vitro matured bovine oocytes were vitrified by the open-pulled straw (OPS) method. In each replicate, a group of oocytes were randomly taken after warming to determine oocyte survival by both morphological evaluation and propidium iodide vital staining. The remainders were evaluated by morphological criterion. Morphologically intact oocytes were co-incubated with frozen-thawed spermatozoa for subsequent development. In situ examination of DNA breaks in oocytes and embryos was conducted using a Fluorescein-FragEL DNA fragmentation detection kit. A caspase-3 detection kit was used to detect caspase-3 activity in oocytes and embryos. Most of the oocytes survived cooling and warming processes as assessed by both morphological evaluation and vital stain. During subsequent culture, some degenerating oocytes displayed observable apoptotic morphology, such as cytoplasmic condensation, cytoplasmic fragmentation, and formation of apoptotic bodies. Biochemical markers of apoptosis, such as apoptotic DNA fragmentation and activation of caspases, were detected not only in oocytes having typical apoptotic morphology, but also in oocytes without observable apoptotic morphology. In embryos, positive signals for both biochemical markers were detected in blastomeres. This experiment suggests that cryopreserved bovine oocytes degenerate via apoptosis during subsequent culture.

Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions

Timed embryo transfer (TET) using in vitro produced (IVP) embryos without estrus detection can be used to reduce adverse effects of heat stress on fertility. One limitation is the poor survival of IVP embryos after cryopreservation. Objectives of this study were to confirm beneficial effects of TET on pregnancy rate during heat stress as compared to timed artificial insemination (TAI), and to determine if cryopreservation by vitrification could improve survival of IVP embryos transferred to dairy cattle under heat stress conditions. For vitrified embryos (TET-V), a three-step pre-equilibration procedure was used to vitrify excellent and good quality Day 7 IVP Holstein blastocysts. For fresh IVP embryos (TET-F), Holstein oocytes were matured and fertilized; resultant embryos were cultured in modified KSOM for 7 days using the same method as for production of vitrified embryos. Excellent and good quality blastocysts on Day 7 were transported to the cooperating dairy in a portable incubator. Nonpregnant, lactating Holsteins (n = 155) were treated with GnRH (100 microg, i.m., Day 0), followed 7 days later by prostaglandin F2alpha (PGF2alpha, 25 mg, i.m.) and GnRH (100 microg) on Day 9. Cows in the TAI treatment (n = 68) were inseminated the next day (Day 10) with semen from a single bull that also was used to produce embryos. Cows in the other treatments (n = 33 for TET-F; n = 54 for TET-V) received an embryo on Day 17 (i.e. Day 7 after anticipated ovulation and Day 8 after second GnRH treatment). The proportion of cows that responded to synchronization based on plasma progesterone concentrations on Day 10 and Day 17 was 67.7%. Pregnancy rate for all cows on Day 45 was higher (P < 0.05) in the TET-F treatment than for the TAI and TET-V treatments (19.0 +/- 5.0,6.2 +/- 3.6, and 6.5 +/- 4.1%). For cows responding to synchronization, pregnancy rate was also higher (P < 0.05) for TET-F than for other treatments (26.7 +/- 6.4, 5.0 +/- 4.3, and 7.4 +/- 4.7%). In the TET-F treatment group, cows producing more milk had lower (P < 0.05) pregnancy rates than cows producing less milk. In conclusion, ET of fresh IVP embryos can improve pregnancy rate under heat stress conditions, but pregnancy rate following transfer of vitrified embryos was no better than that following TAI.

TRANSFER OF FRESH AND CRYOPRESERVED IVP BOVINE EMBRYOS : NORMAL CALVING, BIRTH WEIGHT AND GESTATION LENGTHS

In vitro and in vivo developmental competence of fresh and cryopreserved in vitro produced (IVP) bovine embryos was evaluated up to birth. Three experiments were done. The objective in the first experiment was to develop an optimal vitrification procedure for IVP bovine embryos by determining effects of exposure time (2, 5, 10, 20 min) and temperature (4, 22, 27 degrees C) in cryoprotective agents prior to vitrification on their post-thaw viability. The best combination was used in Experiments 2 and 3. In the second experiment, the importance of post-thaw morphologic selection on pregnancy rates was determined by transferring either selected or unselected single embryos. In the third experiment, pregnancy initiation, maintenance and calving results of vitrified embryos were compared with fresh and conventionally frozen embryos. Fetal losses, birth weights, gestation lengths and frequency of dystocia in the third experiment were monitored. The interaction of exposure time and temperature on both post-thaw re-expansion and hatching rates was significant (P < 0.01). Five minute exposure at 27 degrees C was optimal. In the second experiment, post-thaw selected vitrified embryos had higher pregnancy rates than unselected embryos (P < 0.05). In the third experiment, the pregnancy rate of vitrified embryos did not differ from that of fresh embryos (P > 0.05). However, pregnancy rate of conventionally frozen embryos was lower than that of fresh or vitrified embryos (P < 0.05). Of 92 calves born, 53 were male and 39 were female. Birth weights and dystocia scores of single-born calves did not differ between sexes (P > 0.05). Twin-born calves were lighter than single-born calves (P < 0.05). Overall, the data demonstrate that the transfer of vitrified IVP bovine embryos can result in healthy, apparently normal calves similar to those derived from transfer of fresh and conventionally frozen IVP bovine embryos.

Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos.

Data on biopsied, sexed and cryopreserved in vitro produced (IVP) bovine embryos, and their in vivo developmental competence are very limited. Two preliminary studies were conducted before the primary study. In Experiment 1, post-thaw in vitro developmental competence of biopsied and vitrified IVP embryos was evaluated using re-expansion as an endpoint. In Experiment 2, the pregnancy rates of biopsied fresh, frozen or vitrified embryos following single embryo transfer were compared. Since vitrified embryos resulted in a higher pregnancy rate than frozen-thawed embryos, in the primary study (Experiment 3), all IVP embryos were vitrified following biopsy and sexing (by DNA fingerprinting). In Experiment 3, we compared pregnancy initiation and calving results of heifers in the following treatments: 1) artificial insemination (AI); 2) AI plus contralateral transfer of a single embryo (AI + SET); 3) ipsilateral transfer of single embryo (SET); or 4) bilateral transfer of two embryos (DET). Birth weights, gestation lengths and dystocia scores were recorded. In Experiment 1, post-thaw re-expansion rate of biopsied and vitrified embryos was 85% (70/82). In Experiment 2, pregnancy rates (90 d) were 44% (7/16), 23% (3/13), and 50% (7/14) for vitrified, frozen and fresh embryos, respectively (P < 0.10). In Experiment 3, pregnancy rates of AI and SET were 65% (20/31) and 40% (16/40), respectively (P < 0.05). The pregnancy rate of AI + SET was 75% (27/36) with 11 carrying twins, and the pregnancy rate of DET was 72% (26/36) with 10 carrying twins. All AI fetuses were carried to term, but only half the SET fetuses were carried to term. Similar calving rates were observed in the AI + SET and DET groups, 76 and 70%, respectively, of those pregnant at Day 40. Mean birth weight, dystocia score and gestation length of AI calves were not different from those of SET calves. Mean birth weight and dystocia score of single-born calves were greater than those of twin born calves (P < 0.05). These data demonstrate that biopsied IVP bovine embryos can be successfully cryopreserved by vitrification and following post-thaw embryo transfer, acceptable rates of offspring with normal birth weights can be obtained without major calving difficulties.

Cell allocation in bovine embryos cultured in two media under two oxygen concentrations.

Blastocyst development, total cell number and allocation to inner cell mass (ICM) and trophectoderm (TE) lineages was compared among day 9 hatched blastocysts from four culture treatments in a two-factor design. Two modified commercial media (KSOM and SOF) were used in atmospheres with two oxygen concentrations (5% and 20% O2). No significant effect of medium on development was found, but 20% O2 increased hatching (p < 0.05). There were more cells in hatched blastocysts cultured in KSOM than in SOF (181 vs 136, respectively; p < 0.0001); however, ICM/total cell ratio was not affected by medium. There was a trend suggesting that the proportion of cells allocated to ICM was lower in hatched blastocysts cultured under 5% O2 compared with 20% O2 (0.323 vs 0.380, respectively; p < 0.1). No significant interactions between medium type and oxygen concentration were found. These results indicate that culture components used in this study may affect cell proliferation without altering cell allocation, and that oxygen concentration may play a role in allocation of cells to ICM and TE lineages.

Improvement in recovery of embryos/ova using a shallow uterine horn flushing technique in superovulated Holstein heifers

The aim of this study was two-fold: (1). to compare recovery of embryos/ova from superovulated Holstein heifers by flushing the uterine horns through insertion of the catheter very close to the tip of the horn (deep) or just after the uterine bifurcation (shallow) and (2). to evaluate the hormonal and superovulatory response to estradiol benzoate (EB) treatment prior to superovulation. Ten Holstein heifers (12-16 months) underwent two superovulatory treatments in a cross-over design. Heifers were treated with decreasing doses of FSH from Days 8 to 12.5 of a synchronized estrous cycle. At 4 days prior to superovulation, half of the heifers received EB (5mg, i.m.) or served as Controls, followed by the alternative treatment in the subsequent superovulation. At embryo recovery, one uterine horn was flushed with deep ( approximately 7 cm caudal to the tip of the horn) and the other with shallow ( approximately 5 cm cranial to the beginning of the uterine bifurcation) flushing techniques. Embryos/ova were recovered, counted, and scored. Number of ovulations was estimated by ultrasound. Pretreatment with EB reduced circulating FSH and regressed the first wave dominant follicle with no change in number of large follicles, number of ovulations, number of embryos/ova recovered, or number of transferable embryos. The shallow flushing technique was superior to the deep technique for number of embryos/ova recovered per horn (5.4+/-1.1 versus 3.9+/-0.8) or percentage of embryos/ova recovered per CL (63.9+/-8.6% versus 37.4+/-6.5%). Thus, flushing the entire uterine horn increased recovery of embryos/ova.

Post-thaw survival and pregnancy rates of biopsied, sexed and vitrified bovine IVF embryos

S FOR POSTER PRESENTATIONS POST-THAW SURVIVAL AND PREGNANCY RATES OF BIOPSIED, SEXED AND VITRIFIED BOVINE IVF EMBRYOS Y. Agca, R.L. Monson, D.L. Northey, D.M. Schaefer and J.J. Rutledge Department of Meat and Animal Sciences, University of Wisconsin Madison, Wisconsin 53706, USA Frozen storage of embryos following biopsy and molecular genetic characterization would greatly faciliate assisted reproductive techniques, genetic engineering, and commercial animal production. However, multiple interventions such as handling, biopsying, breaching of zonae and freezing lead to extremely low post-thaw embryo survival (Picard er al., 1985 Vet. Rec. 117: 603-608). Objectives of our first experiment were to determine post-thaw survival rates of vitrified biopsied bovine embryos produced in vitro (IVP) and to compare pregnancy rates of biopsied IVP bovine embryos that were vitrified, conventionally frozen and fresh. In the second experiment, we compared pregnancy initiation and maintenance in recipients that were 1) artificially inseminated (AI), 2) AI plus contralateral transfer of a single embryo (AI+S), 3) transfer of a single embryo (S) and 4) bilateral transfer of twin embryos (double). All transferred embryos were biopsied, sexed female and vitrified in experiment II. Bovine oocytes aspirated from ovaries obtained from an abattoir were matured, fertilized and cultured as previously described (Saeki et al., 1991 Biol.Reprod., 44: 256-260); (Parrish et al 1986, Theriogenology 25: 591-600) Rosenkrans and First, 1994, J. Ani. Sci 72: 434-437). Embryos were mainly Holstein x Angus crosses using semen from a single Angus bull. On day 4 or day 5 of culture (fertilization = day 0) the culture medium was supplemented with 10% heattreated fetal calf serum. Embryos were morphologically evaluated late day 5 or early day 6 and 2 or 3 blastomeres were taken from each embryo selected for biopsy. Biopsied embryos that formed blastocysts by day 7 were selected for further study. Methods for vitrification were modified from (Massip et al., 1989, Animal Reprod. Sci. 19: 117-129). Vitrified embryos were vitrified and thawed as described previously (Agca et al., 1994 Theriogenology 41: 154). After removal of cryoprotectant agents, embryos were rinsed in PBS+lS% FCS prior to placement in culture medium for re-expansion studies or for transfer to recipients heifers. Hatching rate was evaluated over a period of 72 h. All transfers were done using synchronized day 6 or 7 (estrus = Day 0) Hereford x Holstein heifers. Equilibrium freezing and thawing practices were followed (Voelkel et al., 1992, Theriogenology 37: 23-35). Embryo sex determination in experiment II was done via PCR as previously described (Kirkpatrick and Monson, 1993, J. Reprod. Fertil., 98: 335-340) and AI was done at 12 h after onset of estrus using the same bull as was used in generating IVP-embryos. Pregnancies were evaluated by ultrasonographic observation of fetal heartbeats and by rectal palpation at day 35 and 100 post-estrus. Post-thaw re-expansion and hatching rates of biopsied and vitrified embryos were 73% (60/82) and 61% (50/82), respectively. Pregnancy rates were 44% (7/16), 23% (3/13) and 50% (704) for vitrified, slowly frozen and fresh embryos, respectively (p>O.lO) in experiment I. Table 1. Comparison of pregnancy rates of artificially inseminated and/or in-vitro produced, biopsied, sexed and vitrified bovine embryos following embryo transfer (Exp. II) Treatment Preg rate (%) 35 day AI 20/3 1 (65) a AI+ET 27136 (75) a Single ET 16/40 (40) b Twin ET 26136 (72) a Preg rate (%) 100 day 20131 (65) b 21/36 (58) b

CATTLE BLASTOCYST FORMATION ALTERED BY PRESENCE OF FETAL CALF SERUM

Fetal calf serum (FCS) in embryo culture media in domestic animals may cause decreased pregnancy and calving rates and increased calf abnormalities. Yet FCS added during in vitro production of cattle embryos is also associated with an increase in blastocysts produced. This work shows that varying the timing of FCS addition causes a shift in timing of blastocoele formation in cattle embryos. Abattoir-derived oocytes were matured in TC199 with LH, FSH and 10% FCS. After IV]F in TALP using frozen-thawed semen, zygotes were cleaned of cumulus cells and transferred into CRlaa. FCS was added (10% final concentration) directly into embryo cultures on d 4 to 5, d 6 or not at all. Starting on d 6 (IVF = day 0), cultures were examined at 24 h intervals for blastocysts which were removed to CRlaa containing 10% FCS and observed for hatching.