2018: EMBRYO TRANSFER AS A REPRODUCTIVE MANAGEMENT TOOL Embryo transfer as a reproductive management tool Peter J. Hansen Department of Animal Sciences, University of Florida, Gainesville FL Introduction – embryo transfer is more than just a tool for genetic selection Regardless of whether generated by superovulation (otherwise called multiple ovulation – embryo transfer; MOET) or by in vitro production (IVP), the most common purpose of transfer of cattle embryos today is for genetic improvement. The advent of genomics means that genetically-superior females can be identified with acceptable accuracy and these females can be used to produce up to 100 or more embryos from a cow or heifer each year. Embryo transfer can also be used to rapidly establish a herd of high-producing animals, as exemplified by the large numbers of embryos exported from North America to other countries to establish high-yielding dairy herds. Production of embryos in vitro is also an effective means of utilizing sexed semen because one straw of sexed semen can be used to produce multiple embryos in vitro. The pregnancy rates following transfer into recipients is similar between cows receiving embryos produced sexed semen and those receiving embryos produced with conventional semen (Xu et al., 2006; Rasmussen et al., 2012). A less well appreciated use of the bovine embryo is as a tool for improving pregnancy rates. In general, the percent of cows pregnant after receiving an embryo is not appreciably higher than pregnancy success following artificial insemination (AI). However, embryo transfer can improve fertility of cow populations when reproduction is suboptimal because of heat stress. There is also some evidence in dairy cattle that embryo transfer can improve pregnancy rates of lactating cows classified as repeat breeders. The physiological basis for improvement in fertility using embryo transfer Pregnancy rates can be improved in infertile cows by embryo transfer because the procedure bypasses certain causes of pregnancy failure that occur before the time when an embryo is typically transferred into cows (day 7-8 after ovulation). Among the reasons why a cow fails to become pregnant after AI are anovulation, fertilization failure and early embryonic death. For an embryo transfer recipient, these causes are reduced because most embryos used for transfer are the product of a successful fertilization (with the exception of a few parthenotes) and the embryo has been capable of developing to the morula stage or blastocyst stage when it is typically transferred. Embryo transfer during heat stress The best documented example of the use of embryo transfer to improve fertility is for the heat- stressed cow. Much of the negative effect of heat stress on pregnancy establishment involves negative effects on the oocyte or the developing embryo. The oocyte can be damaged by heat stress as early as 105 days before ovulation (Torres-Júnior et al., 2008) and remains sensitive to heat stress through oocyte maturation on the day of ovulation (Putney et al., 1989b). Development of the early embryo is also inhibited by heat stress but it becomes resistant to elevated temperature by about the 8-cell stage of development (Hansen, 2013). Experimentally, heat stress at Day 1 after estrus reduced embryonic development but heat stress at Days 3, 5, and 7 had no effect (Ealy et al., 1993). What this means is that the morula and blastocyst stage embryos that are typically transferred into recipients are largely resistant to damage by maternal hyperthermia and subsequent development is unlikely to be compromised by heat stress. The effectiveness of embryo transfer for improving pregnancy rate during heat stress is shown by representative experiments depicted in Figure A. The improvement in fertility with ET as compared to AI has been seen when embryos were produced by MOET (Putney et al., 1989a; Drost et al., 1999; Rodrigues et al., 2004; Vasconcelos et al., 2011) or by IVP (Ambrose et al., 1999; Al-Katanani et al., 2002; Block et al., 2010; Stewart et al., 2011). The poor cryosurvival of IVP embryos means, however, that pregnancy rate was improved during heat stress when IVP embryos were transferred fresh but not when transferred after conventional freezing or vitrification (see data from Drost et al., 1999; Block et al., 2010 and Stewart et al., 2011 presented in Figure A). Clinical Theriogenology • Volume 10, Number 3 • September 2018229 Using embryo transfer, the summer decline in fertility of lactating cows can be largely eliminated. This idea is shown graphically for an experiment in Brazil in Figure 1B (Rodriques et al. (2004) and there are other examples in the literature (Putney et al., 1988; Chebel et al., 2008; Vasconcelos et al., 2011; Ferraz et al., 2016). One important consideration when carrying out embryo transfer during heat stress is that estrous behavior is reduced in cows exposed to heat stress. Accordingly, the technique is usually performed using fixed-time embryo transfer based on one or more ovulation synchronization protocols such as Ovsynch. In such systems, the day of ovulation (i.e., day of insemination for timed AI protocols) is considered day 0 when calculating synchrony with the embryo. Not every cow responds successfully to ovulation synchronization protocols so an embryo is transferred only into those recipients with a functional corpus luteum as determined by rectal palpation or ultrasonic examination. Repeat breeder cows Another possible use for embryo transfer is to improve fertility of the repeat-breeder cow. These animals are defined as those that have not become pregnant after multiple inseminations (usually three). There are three experiments supporting the idea that embryo transfer can increase fertility for these type of animals as compared to fertility after AI (Table). Each of these studies involved small numbers of animals and further work is warranted. Moreover, it is unlikely that embryo transfer will prevent all sources of infertility associated with the repeat-breeding condition. Ribeiro et al. (2016) observed that negative effects of disease incidence (uterine or non-uterine) before breeding on fertility was apparent in cows subjected to either AI or embryo transfer. Can embryo transfer be used to improve pregnancy rates in the absence of infertility? Based on what was stated at the beginning of this paper, one would expect that pregnancy rate following embryo transfer would usually be higher than pregnancy after AI because failure of achieve pregnancy because of oocyte defects, anovulation, misdiagnosis of estrus, errors in insemination, poor semen quality, fertilization failure, and disruptions in early embryonic development would be prevented. Surprisingly, however, there is not usually a fertility advantage for embryo transfer as compared to AI unless there is some underlying cause of infertility such as heat stress or the repeat-breeder condition. This phenomenon is illustrated in the experiment of Rodrigues et al. (2004) depicted in Figure B. Although pregnancy rates were much higher for embryo transfer than AI in the warm months, pregnancy rates were similar for embryo transfer and AI in cool months of the year. Similar results were seen in studies with lactating cows in Wisconsin (Sartori et al., 2006) and Florida including one performed in cool months (Rasmussen et al., 2012) and one using data collected year-round (Ribeiro et al., 2016). The reason why ET does not meet the promise of improving fertility in most situations is not known. It may be that technical limitations in embryo production or transfer limit the expected improvement in pregnancy rate. Clinical Theriogenology • Volume 10, Number 3 • September 2018 230 Figure. Enhancement of pregnancy rates during heat stress using embryo transfer. Data in Panel A represent results from various experiments in the summer in Florida. Abbreviations are as follows: AI: artificial insemination; EG, frozen in ethylene glycol; F, fresh; Gly, frozen in glycerol; IVFET, embryo transfer with an in vitro produced embryo; MOET, multiple ovulation embryo transfer; TAI, timed artificial insemination; TET-IVF, timed embryo transfer with an in vitro produced embryo; VIT, vitrified. The numbers in the graph represent the day of gestation at which pregnancy diagnosis was carried out. Panel B represents data from a commercial dairy in Brazil in which cows were either inseminated or received an embryo produced by superovulation (Rodriques et al., 2004). Asterisks represent months in which pregnancy rate was different between AI and ET. The figure is reproduced from Hansen (2013). Clinical Theriogenology • Volume 10, Number 3 • September 2018231 Table. Improvement in fertility in repeat-breeder dairy cows by embryo transfer (ET) as compared to artificial insemination (AI). Study Location Embryo type Definition of repeat-breeder Treatment Number of animals Pregnancy rate, percent Tanabe et al., 1985 USA- Pennsylvania MOET >3 infertile services AI at estrus 22 50 ET at estrus 23 70 Son et al., 2007 South Korea MOET 3 or more infertile services AI at estrus 27 18.5 Timed AI 13 7.7 Timed ET 13 53.8 Block et al., 2010 USA – Florida IVP > 3 infertile services Timed AI 33 21.1 Timed ET- fresh 25 60.0 Timed ET- vitrified 31 29.0 Conclusion Based on the experiments conducted to date, there is a high degree of confidence that embryo transfer can improve pregnancy rate when heat stress is a factor. Although the evidence is less clear, there may also be an improvement in fertility using embryo transfer for repeat-breeder cows. The decision to use embryo transfer for fertility enhancement depends on an individual cow’s fertility, the expected improvement in pregnancy rate caused by using embryo transfer, the cost of the embryo available for transfer, and the economic value of getting a cow pregnant. For a discussion of the economic value of embryo transfer, see papers by Ribeiro et al. (2012) and Kaniyamattam et al. (2018). References Al-Katanani YM, Drost M, Monson RL, et al: Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions. Theriogenology 2002; 58:171-182. Ambrose JD, Drost M, Monson RL, et al: Efficacy of timed embryo transfer with fresh and frozen in vitro produced embryos to increase pregnancy rates in heat-stressed dairy cattle. J Dairy Sci 1999; 82:2369-2376. Block J, Bonilla L, Hansen PJ: Efficacy of in vitro embryo transfer in lactating dairy cows using fresh or vitrified embryos produced in a novel embryo culture medium. J Dairy Sci 2010; 93:5234-5242. Chebel RC, Demétrio DG, Metzger J: Factors affecting success of embryo collection and transfer in large dairy herds. Theriogenology 2008; 69:98-106. Drost, M, Ambrose JD, Thatcher M-J, et al: Conception rates after artificial insemination or embryo transfer in lactating dairy cows during summer in Florida. Theriogenology 1999; 52:1161-1167. Ealy AD, Drost M, Hansen PJ: Developmental changes in embryonic resistance to adverse effects of maternal heat stress in cows. J. Dairy Sci 1993; 76:2899-2905. Ferraz PA, Burnley C, Karanja J, et al: Factors affecting the success of a large embryo transfer program in Holstein cattle in a commercial herd in the southeast region of the United States. Theriogenology 2016; 86:1834-1841. Hansen PJ: Cellular and molecular basis of therapies to ameliorate effects of heat stress on embryonic development in cattle. Anim Reprod 2013;10:322-333. Kaniyamattam K, Block J, Hansen PJ, et al: Economic and genetic performance of various combinations of in vitro-produced embryo transfers and artificial insemination in a dairy herd. J Dairy Sci 2018;101:1540-1553. Putney DJ, Drost M, Thatcher WW: Influence of summer heat stress on pregnancy rates of lactating dairy cattle following embryo transfer or artificial insemination. Theriogenology 1989a;31:765-778 Putney DJ, Mullins S, Thatcher WW, et al: Embryonic development in superovulated dairy cattle exposed to elevated ambient temperatures between the onset of estrus and insemination. Anim Reprod Sci 1989b;19:37-51. Clinical Theriogenology • Volume 10, Number 3 • September 2018 232 Putney DJ, Thatcher WW, Drost M, et al: Influence of environmental temperature on reproductive performance of bovine embryo donors and recipients in the southwest region of the United States. Theriogenology 1988; 30:905-922. Rasmussen S, Block J, Seidel GE, et al: Pregnancy rates of lactating cows after transfer of in vitro produced embryos using X- sorted sperm. Theriogenology 2012;79:453-461. Ribeiro ES, Galvão KN, Thatcher WW, et al: Economic aspects of applying reproductive technologies to dairy herds. Anim Reprod 2012;9:370-387. Ribeiro ES, Gomes G, Greco LF, et al: Carryover effect of postpartum inflammatory diseases on developmental biology and fertility in lactating dairy cows. J Dairy Sci 2016;99:2201-2220. Rodrigues CA, Ayres H, Reis EL, et al: Artificial insemination and embryo transfer pregnancy rates in high production Holstein breedings under tropical conditions. Proc 15th Int Congr Anim Reprod 204;2:396 (abstr). Sartori R, Gümen A, Guenther JN, et al: Comparison of artificial insemination versus embryo transfer in lactating dairy cows. Theriogenology 2006;65:1311-1321. Son DS, Choe CY, Cho SR, et al: A CIDR-based timed embryo transfer protocol increases the pregnancy rate of lactating repeat breeder dairy cows. J Reprod Dev 2007; 53:1313-1318. Stewart BM, Block J, Morelli P, et al: Efficacy of embryo transfer in lactating dairy cows during summer using fresh or vitrified embryos produced in vitro with sex-sorted semen. J Dairy Sci 2011; 94:3437-3445. Tanabe TY, Hawk HW, Hasler JF: Comparative fertility of normal and repeat-breeding cows as embryo recipients. Theriogenology 1985;23:687-696. Torres-Júnior JR de S, Pires M de FA Pires, de Sá WF, et al: Effect of maternal heat-stress on follicular growth and oocyte competence in Bos indicus cattle. Theriogenology 2008; 69:155-166. Vasconcelos JL, Jardina DT, Sá Filho OG, et al: Comparison of progesterone-based protocols with gonadotropin-releasing hormone or estradiol benzoate for timed artificial insemination or embryo transfer in lactating dairy cows. Theriogenology 2011; 75:1153-1160. Xu J, Guo Z, Su L, et al: Developmental potential of vitrified Holstein cattle embryos fertilized in vitro with sex-sorted sperm. J Dairy Sci 2006; 89:2510-2518. 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