D.L. Northey

Culture of one- and two-cell bovine embryos to the blastocyst stage in the ovine oviduct

The ovine oviduct was evaluated as a culture system for early bovine embryos. One- to two-cell embryos were collected from superovulated heifers killed 36 or 48 h after the onset of estrus, embedded in agar cylinders, and transferred to oviducts ligated at the uterotubal junction. After 5 d (6.5 to 7.0 d after donor estrus), embryos were recovered and evaluated for development to the late morula or blastocyst stage. In Experiment 1, 86 embryos were cultured in 10 ewes in which the onset of estrus was synchronized with that of the donors. Fifty-eight embryos (68%) were recovered; of these, 31 (53%) had continued normal development. In Experiment 2, development in ovariectomized versus intact cyclic ewes was compared. Recovery from ovariectomized ewes (26/39, 67%) did not differ from intact cyclic ewes (26/35, 74%) and the proportion developing normally also did not differ (ovariectomized: 7/26, 27%; intact cyclic: 11/26, 42%). In Experiment 3, embryo development was compared in anestrous versus ovariectomized ewes. Recovery rate (anestrous: 22/43, 51%; ovariectomized: 20/51, 39%) and the proportion developing normally (anestrous: 8/22, 37%; ovariectomized: 9/20, 45%) did not differ between treatments. Developmental competence of oviduct-cultured embryos was tested by transfer to 16 synchronous heifers, of which eight (50%) became pregnant; five delivered calves. Results indicate that the ovine oviduct provides an adequate site for the culture of early bovine embryos.

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.

Spontaneous embryonic death on Days 20 to 40 in heifers.

Transrectal ultrasound examinations were used in nulliparous Holstein heifers to study the association between time of spontaneous embryonic death (cessation of heartbeat) and luteal regression, and to determine the fate of the conceptus after embryonic death. There was no significant difference between nonbred heifers (n = 135) and bred, nonpregnant heifers (embryonic heartbeat never detected, n = 40) for day of onset of luteal regression (means, 17.6 and 17.9, respectively) or for length of interovulatory interval (means, 20.6 and 20.9 days, respectively). Pregnancy was confirmed by detection of an embryonic heartbeat on Day 24 (ovulation = Day 0) or later, or on two consecutive days prior to Day 24; on average, an embryonic heartbeat was detected on Day 22.0 (n = 104). Pregnancy rate on Day 24 was higher (P<0.02) in heifers bred on Day -1 (116/149, 77.8%) than in heifers bred on Day -2 (51/79, 64.6%), and was higher (P<0.05) in heifers with an embryo transferred ipsilateral to the corpus luteum than in heifers with an embryo transferred contralateral to the corpus luteum (3/4 vs 0/5). Embryonic death (lack of embryonic heartbeat following confirmation of pregnancy) and presumptive embryonic death (embryonic heartbeat detected on one day only, prior to Day 24) were detected prior to Day 25 in one and two bred heifers, respectively, and in one and two heifers with an embryo transferred contralateral to the corpus luteum, respectively. In these six heifers, luteal regression preceded, and apparently caused, embryonic death. In seven of eight heifers in which embryonic death was detected between Days 25 and 40, the onset of luteal regression was detected at least 3 d (range, 3 to 42 d) after detection of embryonic death. The incidence of embryonic death on Days 29 to 32 was lower (P<0.02) in heifers bred on Day -1 than in heifers bred on Day -2 (0 of 96 vs 3 of 40, respectively). In heifers in which luteal regression preceded embryonic death, the conceptus was lost rapidly, with minimal evidence of degeneration. In heifers in which embryonic death preceded luteal regression, there was ultrasonic evidence of conceptus degeneration, but conceptus fluid and tissue appeared to be maintained. In all heifers with embryonic death, the conceptus and its breakdown products apparently were eliminated by expulsion through the cervix rather than by resorption.

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.

Developmental potential of bovine oocytes matured in vitro or in vivo

Bovine oocytes matured in vivo or in vitro were evaluated after sperm-oocyte incubation for frequency of sperm penetration, frequency of male pronuclei formation, and embryonic development. The frequency of sperm penetration was not different for in vitro matured oocytes (216/295, 73%) vs. in vivo matured oocytes (119/176, 70%). However, formation of male pronuclei was reduced (p less than 0.05) for oocytes matured in vitro (149/216, 69%) vs. in vivo (104/119, 88%). Early embryonic development was evaluated 48 h after the onset of sperm-egg incubations. In vitro matured and fertilized oocytes failed to develop to the 2-cell stage (3/88, 3%), whereas oocytes matured in vivo showed normal development (23/56, 40%) to the 2- and 4-cell stage. Development to the blastocyst stage was evaluated after 5 days in ovine oviducts (in vivo). Morulae and blastocysts were obtained only after in vitro fertilization from oocytes that were in vivo-matured (recovered from oviduct, 14/56, 25%; recovered from follicle, 36/80, 45%). Oocytes that were matured in vitro and fertilized in vitro failed to develop to morulae (0/33) in vivo.

Early cycle FSH-p priming as a prelude to superovulating gonadotropin administration in ewes and heifers

The effect of an exogenous FSH treatment in the periovulatory, post-LH surge period on superovulatory response in the subsequent cycle of ewes and heifers was investigated. Thirty-five ewes were synchronized with progestagen pessaries and pregnant mares serum gonadotropin. The day following the onset of estrus (Day 1) 17 ewes received one intramuscular injection of 5 mg follicle stimulating hormone of porcine origin (FSH-p). All 35 ewes received another progestagen pessary on Day 1 and were superovulated with horse anterior pituitary extract (HAP). The ewes were bred and embryos collected 6 days following the onset of estrus. Early cycle FSH-p administration did not increase the subsequent ovulation rate (6.5 vs. 8.4 for controls, n.s.). Recovery rate for the FSH-p treated animals was higher (78.5% vs. 49.3%; P<0.05) as was fertilization rate (100% vs. 62.4%; P<0.05). The final result was a mean of 4.4 transferable embryos per ewe treated among the FSH-p boosted ewes and 2.6 transferable embryos per ewe treated among the control ewes. Twenty-nine heifers were brought into estrus with one 500-μg injection of prostaglandin F2α (PG). Twelve of the 29 heifers were given one intramuscular injection of 10 mg FSH-p on either Day 2 or 3 (Day 1 is the day following the onset of estrus). All heifers were superovulated starting on Day 11–16, over a 4-day period using a decreasing dosage of FSH-p. Prostaglandin was administered at the time of the fifth superovulatory FSH-p injection and the heifers were bred by artificial insemination. Ova were recovered between 2 and 4.5 days following the onset of estrus. There was no effect on ovulation rate due to the interval from FSH-p priming to the day of superovulatory FSH-p initiation. The proportion of heifers that ovulated when given a FSH-p injection early in the cycle was higher than in the control group (94% vs. 68%; P<0.05). The primed heifers had a higher number of ovulations than did the control heifers (16.3 vs. 6.2; P<0.01). The effect of higher ovulation rate carried through all parameters measured, so that the FSH-p primed heifers also had a higher number of fertilized ova than the controls (10.7 vs. 3.9; P<0.05), indicating that there was no significant deterioration in ovum quality due to the FSH-p priming. The results show that FSH-p improved superovulatory efficiency in both sheep and cattle.

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

Nuclear transplantation in bovine embryos

This study was conducted to develop a method for transplanting nuclei in bovine embryos and to test the development of several stages of donor nuclei transplanted to enucleated pronuclear recipient embryos. Pronuclear embryos were centrifuged to reveal nuclei. Nuclei were removed without penetrating the plasma membrane as membrane-bound karyoplasts, and were inserted into enucleated zygotes by electrically induced cell fusion. The highest rate of fusion (79%) occurred in Zimmerman Cell Fusion medium at 100 V for 20 to 40 microseconds with the fusion membranes oriented parallel to the electrodes. The effect of nuclear transplantation on development was tested in pronuclear embryos in which nuclei were removed and reinserted and the embryos were then transferred to sheep oviducts for 5 d. Of the intact nuclear transplant embryos recovered, 5/29 (17%) developed to morulae or blastocysts compared with 11/30 (37%) of the non-manipulated embryos. Two nuclear transplant embryos were transferred to a recipient cow, and both developed to normal offspring. When nuclei from two-, four-, or eight-cell embryos were transplanted to pronuclear recipient embryos, no development was observed.

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.