In the past 25 years, there has been a vast expansion in the development of assisted reproduction techniques for the horse. This review focuses on the integration of these new techniques into the horse industry. Procedures such as embryo transfer, oocyte transfer and intracytoplasmic sperm injection are widely used, but other techniques, such as sexed semen and preimplantation genetic diagnosis, have been slower to be adopted.

Early developmental stages of equine embryos produced in vivo are reviewed and compared with embryo development in a laboratory, including gross similarities and differences. The timing of equine embryo development and morphology in vivo, as well as quality grading systems in other species, are used to introduce potential parameters for assessing equine embryo quality in vitro.

The current status of sex-sorted, refrozen and lyophilised spermatozoa used in equine intracytoplasmic sperm injection (ICSI) breeding programs is reviewed. New semen-handling technologies are being developed that will support and enhance the efficacy of sex-sorted, refrozen or lyophilised stallion spermatozoa in commercial equine ICSI programs.

At fertilisation, spermatozoa activate the oocyte, probably through the introduction of sperm-associated proteins, such as phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We demonstrate that PLCZ1 and PAWP/WBP2NL expression is correlated in stallion frozen–thawed spermatozoa, with a wide variation in PLCZ1 content among stallions. Injection of equine spermatozoa with high versus low PLCZ1 content into bovine or equine oocytes resulted in significantly higher embryo development rates, confirming the importance of PLCZ1 for fertilisation success.

In the horse, the array of successful fertility interventions, pivotal to expanding the offspring pool from mares of high genetic merit, is limited. Therefore it is essential that available treatments are optimised. This study describes an in-depth characterisation of functional parameters of equine female gametes that can be used to improve the success of making ‘test-tube foals’.

Intracytoplasmic sperm injection is now a widespread technology used in the equine breeding industry. In order to improve embryo production, this study describes the nuclear maturation dynamics of equine oocytes. In this study, oocytes that matured early showed an improved blastocyst rate. Furthermore, oocytes that matured late also produced potentially viable blastocysts, although at a lower rate.

Intracytoplasmic sperm injection (ICSI) is used for equine assisted fertilisation. In this retrospective study, oocyte and early embryo morphology and donor age were compared with ICSI success. Pregnancy outcomes were negatively affected by increasing mare age and performance or other stress factors. Faster-developing embryos were more likely to produce pregnancies. Donor factors were more predictive than morphology for ICSI outcome.

Some basic parameters for equine embryo culture are not yet established. We found that blastocyst development after intracytoplasmic sperm injection was not affected by incubator temperature between 37.2°C and 38.2°C, but was reduced under high CO₂ tension (7%), associated with lower medium pH (7.29), during cleavage stage culture. These findings will help optimise the culture environment for in vitro equine embryo production.

In vitro production (IVP) of horse embryos is increasingly popular, but IVP pregnancies more often fail to reach term. We demonstrate that equine IVP embryos are more prone to micronucleus formation, and that slowly developing IVP embryos are less likely to yield a pregnancy. These findings indicate that current embryo culture conditions are suboptimal, and that the speed of embryo development is correlated with pregnancy survival.

A major challenge in the in vitro production of equine embryos is selecting those most likely to result in normal term pregnancy. Here, time-lapse image analysis was used to determine optimal cell division timing in equine embryos and the imaging parameters predictive of developmental success. Our findings provide a non-invasive approach for assessing equine embryo quality in future studies.

In the horse, analysis of morphokinetics using time-lapse imaging and its role in predicting developmental potential has received little attention to date. Associations between morphokinetics, blastocyst development and ability to establish pregnancy were evaluated, and we found that faster in vitro development of equine embryos was associated with increased developmental potential, as in other species studied. Time-lapse imaging was also clinically useful in blastocyst identification.

In vitro embryo production is an important equine reproductive technology. Using time-lapse monitoring, we defined the kinetics of in vitro equine blastocyst development and found that holding oocytes at 25°C for 2 days decreased blastocyst production, whereas holding them at 15°C affected embryo morphokinetics, but not the blastocyst rate. Defining morphokinetic parameters via time-lapse monitoring will allow more precise evaluation of the effects of embryo treatment.

Non-invasive time-lapse microscopy is a means to monitor embryo development without disturbing the culture conditions. The aim of this study was to determine whether horse embryos that succeeded in developing to blastocysts showed different mitotic characteristics to embryos that failed to develop. We determined that embryos that failed to develop to blastocysts exhibited delayed development at several critical early stages.

Cloning is an expensive and inefficient process, but has gained particular interest in the equine industry. In this work we explored different strategies to improve cloning efficiency and we produced the first cloned foal born in Australia. Our data serves as a starting point for the establishment of future protocols for improving equine cloning efficiency.

Equine in vitro embryo production (IVEP) has become increasingly popular in the past decade, but the primary factors involved in the outcome of IVEP (blastocysts per injected oocyte) have not been clearly identified. Although both mare and stallion affect the success of IVEP, mare identity and the number of oocytes recovered per follicular aspiration session appear to be the most reliable predictors of success.

In vitro-produced horse blastocysts exhibit a narrow window of acceptable recipient mare synchrony, and survive best in a Day 4 uterus. in vivo-derived Day 8 embryos are more tolerant, and survive equally well in Day 4–9 recipients. However, survival of in vivo embryos is better in recipient mares that have a long (>5 days) pretransfer oestrus.

Embryos produced by intracytoplasmic sperm injection (ICSI) are typically transferred into recipient mares even when oocytes are collected from a fertile donor. We transferred ICSI-produced embryos back into oocyte donors’ uteri during the same cycle to produce pregnancies, which would eliminate the need for a recipient mare and allow the oocyte donor to carry her own conceptus to term.

In some horse sports, horses of one sex are desirable, whereas those of the other sex have little economic value. We evaluated use of embryo biopsy and sexing via polymerase chain reaction in an active embryo transfer program; biopsy did not affect viability and increased the proportion of female pregnancies to 78%. This technique may decrease both financial and animal loss.

Recently, the demand for in vitro embryo production in the horse has increased worldwide. Most clinical transvaginal ultrasound guided ovum pick-up (OPU) procedures are performed in non-pregnant donor mares, and few experimental studies have described in vitro embryo production from oocytes of pregnant donors 21–150 days in gestation. This report discusses OPU, follicular growth and in vitro embryo production in a pregnant mare during late gestation.