The incompletely fulfilled promise of embryo transfer in cattle-why aren't pregnancy rates greater and what can we do about it?

Abstract

Typically, bovine embryos are transferred into recipient females about day 7 after estrus or anticipated ovulation, when the embryo has reached the blastocyst stage of development. All the biological and technical causes for failure of a female to produce a blastocyst 7 d after natural or artificial insemination (AI) are avoided when a blastocyst-stage embryo is transferred into the female. It is reasonable to expect, therefore, that pregnancy success would be higher for embryo transfer (ET) recipients than for inseminated females. This expectation is not usually met unless the recipient is exposed to heat stress or is classified as a repeat-breeder female. Rather, pregnancy success is generally similar for ET and AI. The implication is that either one or more of the technical aspects of ET have not yet been optimized or that underlying female fertility that causes an embryo to die before day 7 also causes it to die later in pregnancy. Improvements in pregnancy success after ET will depend upon making a better embryo, improving uterine receptivity, and forging new tools for production and transfer of embryos. Key to accelerating progress in improving pregnancy rates will be the identification of phenotypes or phenomes that allow the prediction of embryo competence for survival and maternal capacity to support embryonic development.

Keywords: cattle; embryo transfer; fertility; in vitro fertilization.

Figure 1.

Requirements for a successful pregnancy prone to failure including those that can be alleviated by ET and those that cannot. Birth of a live, healthy calf requires ovulation of an oocyte capable of being fertilized and supporting the development of the resultant embryo (1), deposition of sperm in the reproductive tract capable of fertilizing the oocyte (2), formation of an embryo with the genetic and nongenetic inheritance from the oocyte and sperm that allow it to develop to term (3), and a reproductive tract competent to support gamete transport, fertilization, and development of the conceptus to term (4). For any given mating opportunity experienced by a female, one or more of these requirements can be lacking so that pregnancy is either not established or subsequently fails. Transfer of an embryo into the uterus at ~ day 7 of development can eliminate pregnancy failure caused by problems leading to fertilization failure or early embryonic mortality, including those caused by intrinsic defects in the gametes or embryo (5) as well as an inadequate reproductive tract (6). Transfer of an embryo does not prevent pregnancy losses caused by the inherent inability of the transferred embryo for development to term (7) or by the inability of the reproductive tract to support development after day 7 (8).

Figure 2.

Effect of type of female and reproductive technique (ET using a fresh embryo vs. AI) on the percent of females eligible for pregnancy at ovulation (day 0) and percent pregnant at fertilization (day 1) and blastocyst formation (day 6 to 8). Data for beef heifers were calculated by summing results from two experiments (Carter et al., 2008; Diskin and Sreenan, 1980) and data for lactating dairy cows are from a compilation of studies by Wiltbank et al. (2016). Other sources of data were Sartori et al. (2002) for dairy heifers and Breuel et al. (1993) for lactating beef cows. Note that the estimate of 100% for pregnancy/ET assumes that only live embryos were transferred and that the embryo was correctly positioned during the transfer process. Values would be lower if some embryos transferred were dead or placed incorrectly.

Figure 3.

Two cases where ET increases pregnancy rate as compared with AI—during heat stress and in repeat-breeder cows. The data for the heat stress experiment are from Stewart et al. (2011) and represent P/AI or P/ET at day 40 after estrus for lactating dairy cows in the summer in Texas that were successfully synchronized using an ovulation synchronization protocol (TAI or TET). Both AI and ET involved the use of X-sorted semen. The total number of cows was 485. Embryos were produced in vitro and either transferred fresh (Fr.) or vitrified (Vit.). The data for the study with repeat-breeder cows are from Rodrigues et al. (2007b) and represent data for cows that were either AI at estrus or received an embryo produced by superovulation 7 d after estrus. The data shown are AI or ET results for the months of June to August (n = 1,518 AI and n = 967 ET) for cows that had been inseminated unsuccessfully at least three times previously. The original pregnancy rate for the AI group (24.8%) has been adjusted for this paper by multiplying by 0.8 to account for the approximately 20% of AI cows without a CL.

Figure 4.

Comparison of pregnancy rates per ET for embryos produced in vivo (red and blue) and in vitro (light red and light blue) and that were transferred fresh (F; red or light red solid bars), frozen (Fz; blue or light blue solid bars), or vitrified (V; light blue hatched bars). The difference in pregnancy/ET between fresh and cryopreserved embryos is shown above each pair of bars. Data are from Numabe et al. (2000), Merton et al. (2007, 2013), Stewart et al. (2011), Sanches et al. (2016) and Ferraz et al. (2016).

Figure 5.

Representation of local effects of the bovine blastocyst on the endometrium at day 7 of pregnancy. Of 205 metabolites examined in uterine flushings from the cranial portion of the uterus ipsilateral to the side of ovulation, 22 differed between pregnant and nonpregnant cows. The lipoxygenase products 12(S) and 15(S)-hydroxyeicosatetraenoic acid (HETE) were higher in pregnant cows and the other 20, including specific phospholipids, acylcarnitines, glycine, and sarcosine, were lower. Expression of one lipoxygenase gene, ALOX12, was increased i n the uterotubal junction, whereas another, ALOX15B, was decreased. The decrease in glycine content of the uterine lumen was consistent with decreased expression of the glycine transporter SLC6A9. Consequences of local actions of the blastocyst on the endometrium for long-term function of the blastocyst remain to be determined. The figure is reproduced from Sponchiado et al. (2019) and is reproduced from Scientific Reports through a Creative Commons Attribution 4.0 International License.

Figure 6.

Results from a report of a new system for direct transfer of frozen embryos without loss in pregnancy success as compared with fresh embryos. Shown are pregnancy data in which heifers received either a fresh or frozen embryo. Numbers at the bottom represent the fraction and percent of cows pregnant after transfer. Embryos were produced in vitro and were cultured with BSA until day 6 and then without protein to day 7. Blastocysts at day 7 were transferred to heifers either fresh or after freezing in a medium using a synthetic protein substitute called CRYO3. Frozen embryos were thawed and then transferred directly without removing cryoprotectants. Results are from Gómez et al. (2020).

Figure 7.

Oviduct-on-a-chip. This device was manufactured by soft lithography using two chambers constructed of polydimethylsiloxane (PDMS) and separated by a porous polycarbonate membrane (a). The basolateral chamber was designed to mimic features of the vasculature including delivery of progesterone and estradiol, while the apical chamber was designed to mimic the oviductal epithelium. Both chambers were perfused independently. Oviductal epithelial cells were grown on the apical side of the porous membrane. A photograph of the device is in (b). Movement of oocytes and embryos were arrested in the apical chamber by trapping pillars shown in (c) and (d). The figure is reproduced from Ferraz et al. (2018) and is reproduced from Nature Communications through a Creative Commons Attribution 4.0 International License.

Figure 8.

Degree of repeatability of differentially expressed genes related to embryo survival. The color code indicates whether the gene was upregulated, downregulated, or not significantly associated with either embryo survival. The figure was modified slightly from Zolini et al. (2020a) and was published with permission of Cell and Tissue Research.