Efficacy of Timed Embryo Transfer with Fresh and Frozen In Vitro Produced Embryos to Increase Pregnancy Rates in Heat-Stressed Dairy Cattle1 J. D. AMBROSE,*,2 M. DROST,† R. L. MONSON,‡ J. J. RUTLEDGE,‡ M. L. LEIBFRIED-RUTLEDGE,§ M-J. THATCHER,† T. KASSA,*,3 M. BINELLI,* P. J. HANSEN,* P. J. CHENOWETH†,4 and W. W. THATCHER*,5 *Department of Dairy and Poultry Sciences, University of Florida, Gainesville †Department of Large Animal Clinical Sciences, University of Florida, Gainesville ‡Department of Animal Sciences, University of Wisconsin, Madison §BOMED Inc., Madison
ABSTRACT Our objective was to determine whether pregnancy rates in heat-stressed dairy cattle could be enhanced by timed embryo transfer of fresh (nonfrozen) or frozenthawed in vitro-derived embryos compared to timed insemination. Ovulation in Holstein cows was synchronized by a GnRH injection followed 7 d later by PGF2α and a second treatment with GnRH 48 h later. Control cows (n = 129) were inseminated 16 h (d 0) after the second GnRH injection. On d 7, a fresh (n = 133) or frozen-thawed (n = 142) in vitro-derived embryo was transferred to cows assigned for timed embryo transfer after categorizing the corpus luteum by palpation per rectum as 3 (excellent), 2 (good or fair), 1 (poor), and 0 (nonpalpable). Response to the synchronization treatment, determined by plasma progesterone concentration (ng/ml) ≤1.5 on d 0 and ≥2.0 on d 7, was 76.2%. Mean plasma progesterone concentration on d 7 increased as the quality of corpus luteum improved from category 0 to 3. Concentrations of progesterone in plasma were elevated (≥2.0 ng/ml) at 21 d in 64.7 (fresh embryo), 40.3 (frozen embryo), and 41.4 ± 0.1% (timed insemination) of cows, respectively. Cows that received a fresh embryo had a greater pregnancy rate at 45 to 52 d than did cows that received a frozen-thawed embryo or timed insemination (14.3 > 4.8, 4.9 ± 2.3%). Body condition (d 0) of cows influenced the pregnancy rate and plasma
Received January 22, 1999. Accepted June 14, 1999. 1 Florida Agricultural Experiment Station Journal Series No. R06887. 2 Present affiliation: Alberta Agriculture, Food and Rural Development, Edmonton, Canada. 3 Present affiliation: Institute of Pathobiology, University of Adis Ababa, Ethiopia. 4 Present affiliation: Department of Veterinary Clinical Sciences, Kansas State University. 5 Reprint requests: E-mail:
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progesterone concentrations. In summary, timed embryo transfer with fresh in vitro-produced embryos in heat-stressed dairy cattle improved pregnancy rate relative to timed insemination. (Key words: heat stress, embryo transfer, timed insemination, pregnancy rate) Abbreviation key: BCS = body condition score, CL = corpus luteum, ET = embryo transfer, IVF = in vitro fertilization, TET = timed embryo transfer, TI = timed insemination. INTRODUCTION Pregnancy rates to artificial insemination in lactating dairy cattle typically decline during hot seasons (14). In Florida, under hot and humid summer conditions, pregnancy rates can drop to less than 10% (5). Embryonic loss associated with maternal heat stress is one of the major causes for this decreased fertility. Bovine embryos are highly susceptible to heat stress during early developmental stages, specifically from d 0 to 3 after estrus (9, 29). However, later stage embryos (i.e., beyond d 3) are less susceptible to heat stressinduced losses (9, 11). Putney et al. (30) found no seasonal variation in pregnancy rate to embryo transfer in a large study conducted in the southern United States. Therefore, transferring embryos to recipients at a stage when they are less susceptible to heat stress (i.e., on d 7) may enhance pregnancy rates during periods of heat stress. This hypothesis has been tested by comparing artificial insemination to embryo transfer (ET) with embryos collected from superovulated cows (8, 28) and in vitro-derived frozen-thawed embryos (8). It was demonstrated that pregnancy rates in summer could be increased significantly by transfer of in vivo-derived, frozen-thawed embryos. However, the pregnancy rate from in vitro-derived, frozen-thawed embryos was not different from that following artificial insemination.
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In vivo embryo production is expensive and labor intensive, and the results are often unpredictable because superovulatory responses vary tremendously among donor cows. On the other hand, production of embryos following in vitro fertilization (IVF) is less expensive and less laborious and allows for the production of a large number of embryos within a relatively short time with minimal semen costs. Therefore, transfer of IVF-derived embryos has practical advantages, particularly in large commercial dairy operations such as those prevalent in Florida. Establishment of pregnancy following embryo transfer, among other factors, depends on the availability of well-synchronized recipients. Since efficiency of estrus detection is poor during summer months (1, 40, 43), selection of recipients becomes a difficult task, making embryo transfer less practical during summer. Synchronization of recipients using the protocol (GnRH, d 0 – PGF2α, d 7 – GnRH, d 9) developed for timed insemination (26, 38) eliminates the need for estrus detection. Efficiency of the timed insemination protocol has been demonstrated under Florida conditions (3, 4, 7, 18, 38) and may therefore be a dependable system for the efficient management of recipients for embryo transfer. In the present study, we used this approach to examine systems of reproductive management of dairy cattle potentially relevant to hot climates or seasons. Primary components of the systems were in vitro production of embryos, both fresh and frozen; their shipment to herd sites remote from the laboratory of production; synchronization of recipients without estrus detection; and the application of timed embryo transfer. As pointed out by Rutledge and Seidel (34), individual technologies may have little value used in isolation, but synergism among such technologies may lead to useful systems of production. The primary objective was to determine whether pregnancy rates in heat-stressed dairy cattle could be enhanced by timed embryo transfer of fresh or frozenthawed IVF-derived embryos compared to timed insemination. MATERIALS AND METHODS Cows Four hundred and four lactating Holstein cows, 50 to 140 DIM, (parity range: 1 to 4) were used. Cows were housed in two barns of a 10,000-cow commercial dairy farm located in Okeechobee County, Florida (27°12′N, 80°46′W). Barns were open-sided with concrete flooring and self-locking stanchions. Cows had free access to an adjacent sod-based area. Barns were equipped with fans and misters for evaporative cooling. Fans were operational 24 h daily for forced ventilation, and misJournal of Dairy Science Vol. 82, No. 11, 1999
ters operated in 15-min (3 min on, 12 min off) cycles between 0800 and 2000 h daily. Cows were milked three times daily and allowed to eat a TMR ad libitum. Feed was replenished immediately following each milking. The study was conducted during the hot, humid months of August to October, 1996, in six replicates (three in each barn). The average temperature (24 h mean) for the season was 25.9°C; peak day-time temperatures exceeded 32.2°C for 25 d during the experimental period (6) and a mean relative humidity of 79%. Body condition scores (BCS) were obtained around d 0 (the day after the second GnRH treatment) on 299 cows [scoring scale 1 to 5 (10)] in 0.25 increments. The mean cumulative conception rates in cows not included in the study but maintained under identical management conditions and inseminated at detected estrus during the corresponding period, were 9.4% (1441 services) and 9.6% (1653 services) for the two barns. Production and Transport of Embryos Embryos for transfer without freezing were produced at the University of Wisconsin-Madison, we used Holstein oocytes obtained from an abattoir (Peck Packing Co., Milwaukee, WI) during August to October. Transport of ovaries, recovery, and maturation of oocytes were as described in Saeki et al. (35, 36). After 20 to 22 h of maturation, frozen-thawed sperm after postthaw separation on a Percoll gradient (21) were used to fertilize the oocytes as described by Parrish et al. (23). Semen from four different bulls were used and assigned randomly for IVF to each batch of oocytes. Sperm and eggs were coincubated for 18 to 20 h. The cumulus cells were then removed and the zygotes placed in CR1aa medium (33) for culture. Heat-treated fetal calf serum (10%) was added to culture drops on day 5 (d 0 = fertilization). Embryos that were removed from in vitro culture conditions and transferred to recipient uteri without freezing will be referred to as “fresh” throughout this paper. For fresh transfers, morula or early blastocyststage embryos were removed from culture on d 6, placed in TL-Hepes (22) without glucose supplemented with 0.22 mM sodium pyruvate, 25 µg of gentamicin/ml, and 10% heat-treated fetal calf serum, in 5 ml of sterile, polystyrene Falcon tubes (Beckton and Dickinson, East Rutherford, NJ). Tubes were sealed with Parafilm, placed in a battery-powered portable incubator (Minitub, Inc., Verona, WI) set at 39°C, and shipped to Florida by counter-to-counter air courier (2 to 3 h transit). From the air terminal, embryos were transported by car to the farm site (1-h transit) and remained in the portable incubator overnight. Embryos for freezing were removed from culture at d 7 as expanded or expanding blastocysts and cryopre-
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served for direct transfer in modified Dulbecco’s PBS containing 1.5 M ethylene glycol and 0.4% BSA following a previously described procedure (42) with the addition of 0.2 M sucrose. Most of the frozen embryos were produced in Wisconsin and air-transported to Florida in one shipment and held in liquid nitrogen, until used. One batch of frozen embryos (38% of total) was produced at the University of Florida following the same culture and freezing procedures described. Synchronization of Ovulation All animals received GnRH (Cystorelin威, Sanofi Animal Health Inc., Overland Park, KS; 100 µg, i.m.) followed 7 d later by PGF2α (Lutalyse威, Pharmacia-Upjohn, Kalamazoo, MI; 25 mg, i.m.) and a second GnRH treatment given again at 48 h after PGF2α. Cows were not observed for estrus but were randomly assigned to timed insemination 16 h after the second injection of GnRH (TI, n = 129) or timed embryo transfer to receive either a fresh (TET-Fresh, n = 133) or frozen-thawed (TET-Frozen, n = 142) embryo. Embryos were transferred approximately 7.5 d after the time of second GnRH injection. Embryo Transfer On the morning of d 7 (d 0 = d of TI in control cows), fresh embryos were removed from the portable incubator and evaluated at 40× under a stereo-zoom microscope. Good-quality embryos (expanded and expanding blastocysts) were packaged individually in straws. The straws were laid horizontally (to prevent loss of embryos due to drifting) in a container, protected from direct light, and carried to the barn for transfer. Prior to embryo transfer, cows were palpated for the presence of a corpus luteum (CL) and for abnormalities of the reproductive tract. Three cows with palpable abnormalities such as pyometra or extensive uterine adhesions were excluded from the study. Based on size and palpable characteristics, CL were categorized as 3 (excellent quality: well-defined large CL with prominent crown, approximate size ≥2.0 cm), 2 (good to fair quality: easily palpable small CL with rounded surface but no prominent crown; ≥1.0 cm and TET frozen and TI control). Recently, Massip et al. (17) reported that the majority of embryonic losses occurred from 21 to 45 d after transfer of in vitro-derived fresh and frozen-thawed bovine embryos. More recent evidence suggests that a majority of embryonic loss occurs before d 42 in heat-stressed cows (41). Several previous studies have shown that a relationship exists between body condition and pregnancy rates (4, 18). Results of the present study detected a 37% increase in pregnancy rate for a whole unit gain in body condition. Similarly, a relationship was detected between body condition and plasma progesterone concentration. These results reemphasize the importance of returning cows to a positive energy state as soon as possible to avoid major losses due to reproductive wastage. In conclusion, transfer of fresh IVF-derived embryos to heat-stressed dairy cattle increased pregnancy rates in the present study. However, transferring frozenthawed IVF-derived embryos was of no advantage. Onfarm transfer of fresh IVF-derived embryos has practical difficulties and requires trained personnel and equipment for the evaluation and packaging of embryos at farm sites. If techniques that allow transportation of fresh embryos prepacked in straws (without the risk of losing embryos) could be developed, transfer of fresh IVF-derived embryos may be realistic under some situa-
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tions. On the other hand, if pregnancy rates comparable to transfer of fresh IVF-derived embryos could be achieved with frozen-thawed IVF-derived embryos, timed embryo transfer to enhance pregnancy rates during summer months may become a practical alternative. Further research is therefore needed to enhance freezability of IVF-derived embryos. Timed embryo transfer without estrus detection proved convenient and practical under field conditions. Even under extreme heat stress conditions, cows in better body condition tend to have increased rates of pregnancy. Since progesterone concentration increases with CL quality, recipient cattle in good body condition and well-defined CL should be used, where possible, to increase the chances of pregnancy under heat stress conditions. ACKNOWLEDGMENTS The authors thank the management of Larson Dairy Inc., Okeechobee, Florida, for providing the animals, drugs, and facilities for the study. The excellent cooperation of the administration and barn staff at Larson Dairy Inc. is deeply appreciated. Special thanks are due to Dragan Momcilovic and Peter Morresey of the College of Veterinary Medicine, and Thais Diaz and Frederico Moreira, Department of Dairy and Poultry Sciences, University of Florida, for their assistance during the experiment. The technical support of Jesse Johnson with progesterone assays is appreciated. Research supported by USDA-BARD Grant no. 94-343391212 and USDA-CBAG Grant no. 95-34135-1860. REFERENCES 1 Abilay, T. A., Johnson, H. D., and M. Madan. 1975. Influence of environmental heat on peripheral plasma progesterone and cortisol during the bovine estrous cycle. J. Anim. Sci. 58:1836– 1840. 2 Al-Katanani, Y., D. W. Webb, and P. J. Hansen. 1998. Factors affecting seasonal variation in non-return rate of lactating dairy cows. J. Anim. Sci. 76 (Suppl 1): 217. (Abstr.) 3 Arechiga, C. F., C. R. Staples, L. R. McDowell, and P. J. Hansen. 1998. Effects of timed insemination and supplemental β-carotene on reproduction and milk yield of dairy cows under heat stress. J. Dairy Sci. 81:390–402. 4 Burke, J. M., R. L. de la Sota, C. A. Risco, C. R. Staples, E. J. Schmitt, and W. W. Thatcher. 1996. Evaluation of timed insemination using a gonadotropin-releasing hormone agonist in lactating dairy cows. J. Dairy Sci. 79:1385–1393. 5 Cavestany, D., A. B. el-Wishy, and R. H. Foote. 1985. Effect of season and high environmental temperature on fertility in Holstein cattle. J. Dairy Sci. 68:1471–1478. 6 Climatological Data, Florida. 1996. United States Environmental Data Service. 100:1–13. 7 De la Sota, R. L., J. M. Burke, C. A. Risco, F. Moreira, M. A. DeLorenzo, and W. W. Thatcher. 1998. Evaluation of timed insemination during summer heat stress in lactating dairy cattle. Theriogenology 49:761–770. 8 Drost, M., J. D. Ambrose, M-J. Thatcher, C. K. Cantrell, K. E. Wolfsdorf, J. F. Hasler, and W. W. Thatcher. Conception rates after artificial insemination or embryo transfer in lactating dairy cows during summer in Florida. Theriogenology (In press).
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