Equine Embryo Development Research Articles

Effect of pre-maturation oocyte timing on equine ICSI embryo development rates

Within the last 5 years, client adoption of ovum-pick-up (OPU) paired with equine intracytoplasmic sperm injection (ICSI) and in vitro embryo production (IVP) in Brazil has been rapid. Equine ICSI has facilitated the use of expensive semen be it imported, rare, poor-quality or low motility for IVP. A pre-maturation step occurs between OPU on the farm and transport to the lab. Oocytes are kept in 5 ml vials of pre-maturation medium, held at 22°C until placement in maturation medium. Oocytes in pre-maturation medium are kept at this low temperature to inhibit the onset of germinal vesicle breakdown and the onset of oocyte maturation. If maturation begins during transport, oocyte maturation rates and oocyte quality would be severely reduced. Recently, (Merlo, B et al 2022 Anim Reprod Sci 107071) reported different rates of equine ICSI embryo development based on timing of oocyte pre-maturation and maturation. In our IVP system, oocyte maturation time is tightly controlled between 28 and 32 hours, so we decided to evaluatethe timing of pre-maturation since it can vary between 0 and 24 hours. This variation is based on the time required for OPU completion by multiple teams, aspirating on the same day in different locations. We tested the potential impact of oocyte pre-maturation in 3-time intervals 0-8, 9-16 and 17-24 hours, in the 2021-2022 breeding season. The average blastocyst formation rate based on total oocytes collected was 17%. Embryo development from oocytes aspirated in the 3 pre-maturation time intervals was: 0-8 hours, 522 embryos produced from 3413 oocytes aspirated (15.3%); 9-16 hours, 471 embryos produced from 2667 oocytes aspirated (17.7%) and 17-24 hours, 3931 embryos produced from 22,896 oocytes aspirated (17.2%). There were no significant differences observed (Mann-Whitney test p >0.05), demonstrating that up to 24 hours, the holding time of pre-maturation oocytes, held at 22.5°C had no significant impact on embryo development in our equine ICSI embryo production system.

Cloning horses by somatic cell nuclear transfer: Effects of oocyte source on development to foaling

The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abattoir-sourced ovaries and from live mares by OPU. A total of 1,128 oocytes were obtained, of which 668 were abattoir-derived and 460 were OPU-derived. The methods used for in vitro maturation and SCNT were identical for both oocyte groups, and the embryos were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham medium supplemented with 10% fetal calf serum. Embryo development in vitro was assessed, and Day 7 blastocysts were transferred to recipient mares. The embryos were transferred fresh when possible, and a cohort of vitrified-thawed OPU-derived blastocysts was also transferred. Pregnancy outcomes were recorded at Days 14, 42 and 90 of gestation and at foaling. The rates of cleavage (68.7 ± 3.9% vs 62.4 ± 4.7%) and development to the blastocyst stage (34.6 ± 3.3% vs 25.6 ± 2.0%) were superior for OPU-derived embryos compared with abattoir-derived embryos (P < 0.05). Following transfer of Day 7 blastocysts to a total of 77 recipient mares, the pregnancy rates at Days 14 and 42 of gestation were 37.7% and 27.3%, respectively. Beyond Day 42, the percentages of recipient mares that still had a viable conceptus at Day 90 (84.6% vs 37.5%) and gave birth to a healthy foal (61.5% vs 12.5%) were greater for the OPU group compared with the abattoir group (P < 0.05). Surprisingly, more favourable pregnancy outcomes were achieved when blastocysts were vitrified for later transfer, probably because the uterine receptivity of the recipient mares was more ideal. A total of 12 cloned foals were born, 9 of which were viable. Given the differences observed between the two oocyte groups, the use of OPU-harvested oocytes for generating cloned foals is clearly advantageous. Continued research is essential to better understand the oocyte deficiencies and increase the efficiency of equine cloning.

Effect of method and media type for in vitro fertilization on equine (Equus ferus caballus) embryo development

Demonstrating the role of in vitro fertilization in the equine family, the current study aimed to monitor the rates of equine in vitro-fertilized and cleaved oocytes based on microdroplet and intracytoplasmic sperm injection (ICSI) methods. The fertilization process was carried out in four main media consisting of Tyrod?s albumin lactate pyruvate and TCM-199 (1:1): F (TCM-199); assigned for the ICSI method, FI (contained 0.01 mg/ml heparin + 0.01 ng/ml hypotaurine), FII (contained 0.3 mg/ml calcium chloride + 0.1 mg/ml magnesium chloride) and FIII (contained 0.05 mg/ml hypotaurine-epinephrine). The results indicated an increase in the fertilization rate by the ICSI method (65.71%) compared to the microdroplet method (39.25%) (p 0.04). The rates of unfertilized and degenerated oocytes in the ICSI method decreased, compared to the microdroplet method, to 8.57% and 25.72%, respectively; p&lt; 0.001. The rate of fertilized oocytes in the FI medium increased to 47.61% (p&lt; 0.05) compared to oocytes of FII and FIII treatments (37.64% and 30.00%, respectively). On in vitro culture, the rate of cleavage increased to 74.62% in the CI medium (SOF) compared to those in the CII medium (DMEM-F12) (61.29%); p &lt;0.001. Zygotes cultured in the CII medium achieved a higher rate of blastocyst formation (30%) compared to those in the CI medium (26.31%); p&lt; 0.001. Applying the ICSI method and SOF culture media led to high yields of equine embryos.

Oocytes obtained by ovum pick-up from live mares as an alternative to abattoir-derived oocytes for the development of equine embryos produced by somatic cell nuclear transfer

The production of equine embryos by somatic cell nuclear transfer (SCNT) is limited by the availability of immature oocytes. The main source of oocytes for SCNT comes from ovaries obtained from slaughter houses. However, in some countries there is no horse abattoirs or their number is limited which precludes the use of SCNT for the production of equine cloned embryos. Ovum pick-up (OPU) is a reproductive assisted technique which is growing rapidly in the equine industry which allows obtaining oocytes from live mares. OPU is mainly used in combination to ICSI (intracytoplasmic sperm injection) for the production of in vitro embryos. Oocytes collected from live recipient mares by OPU could be an alternative for SCNT in countries in which there are limited number of equine slaughter houses. However, it is unknown the effect of the oocytes’ source on the development of equine cloned embryos. The aim of this study was to compare the development of equine SCNT embryos produced using oocytes either retrieved by OPU or recovered from abattoir-sourced ovaries. For the study, a total of 1128 oocytes were used, of which 663 were from abattoir-sourced ovaries and 495 were from live mares programmed for OPU. The harvested ovaries were transported to the lab within 1 h for processing. The methods used for in vitro maturation (IVM), SCNT andembryo culture in vitro were identical for both sources of oocytes. The rates of maturation, cell fusion, cleavage and blastocyst formation at Day 7 were evaluated. Transferrable grade blastocysts were transferred to recipient mares 4 to 5 days after ovulation and pregnancy was diagnosed at Days 14 and 42 by ultrasound. The maturation rate of OPU-derived oocytes was lower than that of abattoir-derived oocytes (50.3 ± 2.7% vs . 61.9 ± 3.4%; P < 0.05). However, the rates of cell fusion (90.7 ± 2.6% vs. 81.9 ± 5.2%), cleavage (68.8 ± 3.9% vs . 61.9 ± 5.0%) and blastocyst formation (34.3 ± 2.8% vs. 25.4 ± 2.1%) were greater (P < 0.05) for OPU-derived embryos compared with abattoir-derived embryos. While similar proportions of OPU- and abattoir-derived blastocysts initiated pregnancy at Day 14, a greater proportion of OPU-derived blastocysts developed to Day 42 of gestation (13 of 50 embryos) compared with abattoir-derived blastocysts (3 of 27 embryos; P < 0.05). The results show that abattoir-sourced oocytes subjected to IVM have the potential to support the development of SCNT embryos that can initiate pregnancy. However, given the observed difference in subsequent in vivo development, the use of OPU-derived oocytes for equine SCNT embryo production is recommended. Further research is needed to improve the IVM of abattoir-sourced oocytes

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism.

Equineembryos exhibit an unusual pattern of glucose tolerance in vitro and are currently cultured in hyperglycaemic conditions. Our main objective was to analyse the effect of different glucose concentrations on in vitro-produced equine embryo development and quality. Experiments comparing in vitro and in vivo produced embryos. Oocytes (n=641) were collected from post-mortem ovaries, matured in vitro and fertilised by intracytoplasmic sperm injection (ICSI). Embryo culture was divided from Day 0 to Day 4 and from Day 4 to Day 9 in three groups: 5-10 (5 and 10mmol/L glucose respectively; n=87); 5-17 (5 and 17.5mmol/L; n=66); and 10-17 (10 and 17.5mmol/L; n=117). A control group of 20 in vivo produced blastocysts was included. Cleavage and blastocyst rates were evaluated and embryos were snap-frozen for analysis of the relative mRNA expression of genes related to mitochondrial function, DNA methylation, apoptosis, glucose transport and metabolism. No differences were observed in the cleavage or blastocyst rates among in vitro groups. Under high glucose conditions in vitro (10-17 group), BAX/BCL2 was higher, and PFKP, LDHA and COX2 were overexpressed compared to all other groups. The two groups with 5mmol/L glucose concentration during the first culture stage (5-10 and 5-17) displayed similar patterns which differed to the 10-17 group. Conclusions related to embryo quality are based on gene expression patterns. Transfer of in vitro-produced embryos would reveal whether the observed differences improve embryo developmental competence. Five mM glucose during the first days of culture seems to be preferable to avoid over-activation of embryonic glycolytic pathways. Further studies are necessary to determine whether this improves embryo developmental competence.

99 Short-term storage of equine embryos at 5 or 20°C does not cause lipid peroxidation

Maintaining the integrity of equine embryos during storage for transportation is essential for successful conception after transfer. During storage, reactive oxygen species may originate from embryo metabolism, causing lipid peroxidation and increasing its end product malondialdehyde (MDA); MDA is one of the important biomarkers for oxidative stress. This study aimed to evaluate the temperature curve, pH and lipid peroxidation of equine embryos stored in holding medium after 24h at 5 or 20°C. Embryos (n=33) were collected on Day 7 (n=21, 7 embryos per group) or Day 8 (n=12) after ovulation and assigned to four groups: Day 7 control (D7, fresh); Day 7, 24h at 5°C (E5C); Day 7, 24h at 20°C (E20C); and Day 8 control (D8, fresh 24h time control). After collection, embryos were washed and kept in holding medium (Minitube) for morphological classification and measurements. For pH and lipid peroxidation measurement, embryos were kept in a fixed volume of holding medium (150µL) within a microtube (200µL); the microtube was kept within a falcon tube (50mL) inside of an Equitainer (Hamilton Biovet). The temperature was recorded by a data logger (Testo 175, Testo) every 10min for 24h. The pH was assessed by a pH meter using a microelectrode (InLab Ultra-Micro-ISM, Mettler Toledo) for small sample volumes. Lipid peroxidation was assessed using the MDA assay kit (catalog number MAK085, Sigma-Aldrich) according to the manufacturer's instructions. Statistical analyses were performed using the Kruskal-Wallis nonparametric test and Mann-Whitney U to compare differences among groups. Embryo size differed (P&amp;lt;0.05) between D7 (383±41 µm) and D8 (1044±131 µm). Storage temperature (E5C or E20C) did not affect embryo size (382±47 and 553±99µm, respectively; P&amp;gt;0.05). The temperature curve was similar (P&amp;gt;0.05) among embryos within the treatment groups during the storage period. The pH (7.22±0.07 and 7.22±0.09) did not differ (P&amp;gt;0.05) between E5C and E20C. Lipid peroxidation levels were below the limit of quantification (0.04 nmol) in all groups. The present findings suggest that holding temperature does not affect the size, pH, or lipid peroxidation of equine embryos stored for 24h in holding medium. However, our previous studies (Gastal et al. 2018 J. Equine Vet. Sci. 66, 185) have shown that holding temperature influences the expression of genes involved in equine embryo development. In conclusion, variations regarding embryo development and conception rate after transfer of embryos stored at different temperatures might be related to factors other than lipid peroxidation.

The mule (Equus mulus) as a recipient of horse (Equus caballus) embryos: Comparative aspects of early pregnancy with mares

The aim of this study was to compare the embryonic and early fetal development of horse embryos between recipient mules and mares from day 10–60 of pregnancy, in addition to hormonal (eCG and progesterone), ovarian, and uterine characteristics for approximately 4 months. Embryo donor mares (n = 5) and two groups of recipients (acyclic mules, n = 7; cyclic mares, n = 7) were used. Donor mares were monitored daily by transrectal ultrasonography and inseminated using fresh semen. Cyclic recipient mares were synchronized with the donor’s ovulation using PGF2α and deslorelin acetate. Mules were prepared for the embryo transfers with estrogen and progestagen. Embryo collection and transfer were performed 8 days after ovulation of the donor mares. Pregnancy diagnosis with ultrasonography began 1 day after embryo transfer. After pregnancy confirmation, the recipient mules received long-acting progesterone once weekly for at least 120 days. The first day of detection (day 10) of an embryonic vesicle (EV) was similar between mules and mares. A period of extensive intrauterine mobility of the embryonic vesicle was observed similarly in mules and mares from days 10–17. The day of fixation of the EV in mules tended to be 1-day earlier than in mares; however, the diameter and growth rate of the EV did not differ between the two species. The embryo proper was first detected at day 20, and the crown-rump, width, and diameter were similar between the two recipient types. The heartbeat and allantoic sac tended to be detected 1 day later in mules than in mares, while the umbilical cord was first observed around day 40 in both species. Besides the expected differences found in ovarian aspects and eCG production, similar endometrial diameter, uterine tone and echotexture, and progesterone levels were seen between the two types of recipients. In conclusion, striking ultrasound similarities in equine embryo and fetal development, and uterine characteristics were seen between mules and mares used as recipients of horse embryos.

Equine non-invasive time-lapse imaging and blastocyst development.

In this study we examined the timeline of mitotic events of invitro-produced equine embryos that progressed to blastocyst stage using non-invasive time-lapse microscopy (TLM). Intracytoplasmic sperm injection (ICSI) embryos were cultured using a self-contained imaging incubator system (Miri®TL; Esco Technologies) that captured brightfield images at 5-min intervals that were then generated into video for retrospective analysis. For all embryos that progressed to the blastocyst stage, the initial event of extrusion of acellular debris preceded all first cleavages and occurred at mean (±s.e.m.) time of 20.0±1.1h after ICSI, whereas 19 of 24 embryos that did not reach the blastocyst stage demonstrated debris extrusion that occurred at 23.8±1.1h, on average 4h longer for this initial premitotic event (P<0.05). Embryos that failed to reach the blastocyst stage demonstrated a 4-h delay compared with those that reached the blastocyst stage to reach the 2-cell stage (P<0.05). All embryos that reached the blastocyst stage expressed pulsation of the blastocyst with visible expansion and contraction at approximate 10-min intervals, or five to six times per hour. Using a logit probability method, we determined that 2- and 8-cell stage embryos could reasonably predict which embryos progressed to the blastocyst stage. Together, the results indicate that TLM for equine embryo development is a dynamic tool with promise for predicting successful embryo development.

Equine Embryo and Early Fetus Development in Recipient Mules and Mares

Equine Embryo and Early Fetus Development in Recipient Mules and Mares

Advances in equine cloning by somatic cell nuclear transfer (SCNT) technique in horses: a review

Cloning a horse means using the genetic material (DNA) from a donor horse to produce a genetically identical foal. This technique involves collecting the DNA from the donor and inserting that DNA into an egg from another mare whose, DNA content has been removed, fusing donor nucleus with enucleated recipient oocytes, which then develops as an embryo, in vitro culture of embryo and lastly transfer cultured embryo into the uterus of a recipient mare. The modification of the in vitro culture conditions which can be suitable for equine oocyte activation, oocyte maturation and embryo development are the fundamental steps for a successful in vitro procedure for somatic cell nuclear transfer (SCNT) in the horse to avoid the embryo losses. In general, few studies are available in the literature on equine in vitro embryo production and it is only recently that reports have been published on completely in vitro production of equine preimplantation embryos by means of in vitro oocyte maturation The present review discusses the latest developments in the field of equine cloning technique with the employment of SCNT. The basic understanding of SCNT for in vitro culture conditions is relevant to the increased efficiency of cloning. The available genotype can be used by SCNT which can enhance the vigour of a particular infertile or low fertile animal to produce normal fertility.

The effects of preovulatory administration of oral letrozole on ovulatory follicle and the subsequent corpus luteum in mares

Progesterone acts as an important factor in maintenance of pregnancy and development of equine embryo. Treatment with letrozole, a nonsteroidal inhibitor of aromatase enzyme regulating the synthesis of estrogens, during the preovulatory period has been reported to result in larger corpus lutea (CLs) with greater progesterone production in human and bovine. The present study was conducted to evaluate the effect of preovulatory administration of letrozole on the size of CL and progesterone concentration. In the present study, mares (n = 7) received PGF2α (250 μg) 14 days after the observed ovulation and were subjected to daily ultrasonography to evaluate the development of the largest follicle and the resultant CL for two consecutive estrous cycles. In addition, progesterone concentration was measured over the course of luteal phases. During the first cycles, as control cycles, mares received no treatment. During the second cycles, mares received letrozole (0.5 mg/kg) orally beginning from PGF2α administration for 3 days. Letrozole reduced the diameter of ovulatory follicle (P < 0.05) and advanced the time of ovulation (P < 0.05). There was also a tendency for formation of smaller CLs following letrozole administration (P = 0.065). Moreover, concentration of progesterone during midluteal phase was lower following letrozole treatment (P < 0.05). In conclusion, the present study showed that letrozole treatment during the preovulatory period could result in premature ovulation and impair the function of subsequent CL.

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.

The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals.

Influence of semen type on equine embryo development after ICSI in a commercial program

Influence of semen type on equine embryo development after ICSI in a commercial program

Effect of medium variations (zinc supplementation during oocyte maturation, perifertilization pH, and embryo culture protein source) on equine embryo development after intracytoplasmic sperm injection

Prospective studies were conducted to help define procedural factors affecting in vitro embryo production via intracytoplasmic sperm injection (ICSI) of equine oocytes. In experiment 1, use of 10% fetal bovine serum as a protein source in embryo culture medium resulted in a higher blastocyst rate than did use of a combination of 3% fetal bovine serum, 3% equine preovulatory follicular fluid, and 4% human serum substitute (37% vs. 15%, respectively, P < 0.05). In experiment 2, the effect of zinc supplementation (0, 0.5, 1, or 1.5 μg/mL) during IVM was examined. There were no significant differences in rates of cleavage or blastocyst development (20%–31%). However, the proportion of blastocysts that developed on Day 7 for the added-zinc treatments was significantly higher than that for the control treatment (45% vs. 8%). In experiment 3, we tested whether use of high-pH medium (pH 8.0–8.4) during ICSI procedures would improve blastocyst rate when sperm with low cleavage rates after ICSI was used. When high-pH conditions were used for sperm preparation and also for the first 2 hours of incubation of injected oocytes after ICSI, the cleavage rate was unaffected but no blastocysts developed (0% vs. 24% for control). When high-pH conditions were used for sperm preparation only, the blastocyst rate was 37%. This was repeated using sperm from a second stallion; there was no significant difference in cleavage or blastocyst rates between sperm preparation in high pH vs. control medium. These findings add to our knowledge of factors affecting in vitro production of equine embryos.

Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse

The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes from domestic animals tested in our study, the feline ooplasm might be the most appropriate recipient to partially allow preimplantation embryo development of iSCNT equine embryos.

The effects of embryo-uterine asynchrony on early embryonic development

Equine pregnancy has an extraordinary ability to tolerate embryo-uterine asynchrony. Indeed, while ruminant embryos will not survive negative asynchrony (i.e. the recipient ovulated after the donor) of more than 2 days [Lawson RAS,et.al, J Reprod Fertil, 1975;45:101-7], horse embryos will continue to develop in the uterus of a recipient that ovulated up to 7 days later [Wilsher S,et.al, Reproduction 2010;139:575-85.]. However, negative asynchrony of more than 4 days appears to compromise embryo development and survival and may therefore be a useful model to study embryo-maternal communication. This study examined the effects of negative uterine asynchrony on the development of equine embryos over a period of either 6 or 11 days. Embryos were recovered on day 8 after ovulation and transferred non-surgically to synchronous (day 8; donor-recipient difference 1⁄4 0 days) or asynchronous (day 3; donor-recipient difference 1⁄4 -5 days) recipient mares. The resulting conceptuses were recovered either 6 days later by uterine lavage or 11 days later using a video-endoscopically-guided net (n 1⁄4 5 per group in all cases). Prior to recovery, vesicle size was determined ultrasonographically; following recovery the embryonic disc and, for conceptuses recovered on day 11 post-ET, the embryo proper were identified, measured and staged in terms of primitive steak formation and indices of embryo development such as crown-rump length and somite

34 EFFECT OF DONOR CELLS SERUM STARVATION ON THE DEVELOPMENT OF AGGREGATED ZONA FREE CLONED EQUINE EMBRYOS

Donor cell synchronization for nuclear transfer (NT) is one of the crucial steps in the cloning procedure, and it has been shown that different methods affect embryo development. The goal of the present study was to determine the effect of serum starvation in combination with growth to confluence of the somatic donor cells, on in vitro embryo development and quality of aggregated cloned equine embryos. Oocyte collection, maturation, cloning, and activation procedures were performed as described previously by (Gambini et al. 2012 Biol. Reprod. 87, 15). Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of 2 horses. They were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics. After cell multiplication, they were cryopreserved and stored in liquid nitrogen. After thawing, 2 groups of cell synchronization were established. Group I: growth to confluence for 3 to 5 days before NT followed by serum starvation in DMEM supplemented with 0.5% FBS for 24 h prior to NT; group II: growth to confluence for 3 to 5 days before NT. After activation, reconstructed embryos (RE) were cultured in SOF in the well of well system, placing 3 RE per well. Cleavage and blastocyst formation at Day 7 to 8 were assessed. At Day 7 to 8, embryos were measured and some of each group were fixed with paraformaldehyde to measure DNA fragmentation through the DeadEnd fluorometric TUNEL system (Promega, Madison, WI, USA). In vitro embryo development, on a per embryo and cleaved RE basis, and blastocyst size was compared using the chi-squared test; the proportion test was used for statistical analysis of DNA fragmentation levels (fragmented DNA cells/total cells). There were no statistic differences on cleavage per RE (I: 136/177, 80.2%; II: 142/171, 79.6%), blastocyst rates at Day 7 (I: 29/58, 49.1%, II: 22/56, 39.2%), or on blastocyst size at Day 8. Statistical differences were observed in blastocyst rate at Day 8 (I: 42/58, 72.4%, II: 30/56, 53.5%) and in blastocyst DNA fragmentation levels (I: 202/2464, 8%; II: 173/1440 12%). In conclusion, the restriction of FBS to the cell culture medium 24 h before cloning seams to improve embryo development at Day 8 and also reduces the level of apoptosis in cloned blastocyst, suggesting a better embryo quality. For these reasons, we consider that the addition of the restriction of FBS to the growth to confluence is beneficial for cloned equine embryo development.

Equine Cloning: In Vitro and In Vivo Development of Aggregated Embryos1

The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.

Factors affecting equine embryo development after post-mortem ICSI in a clinical program

Factors affecting equine embryo development after post-mortem ICSI in a clinical program

Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos

Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regulated gene was examined by qPCR in equine in vivo embryos from morula to expanded blastocyst stage. SRP14, RPL4 and PGK1 were identified by geNorm analysis as stably expressed reference genes suitable for data normalisation. RPL13A expression was less stable and changed significantly during the period of development examined, rendering it unsuitable as a reference gene. As anticipated, CDX2 expression increased significantly during embryo development, supporting its possible role in trophectoderm specification in the horse. In summary, it was demonstrated that evidence-based selection of potential reference genes can reduce the number needed to validate stable expression in an experimental system; this is particularly useful when dealing with tissues that yield small amounts of mRNA. SRP14, RPL4 and PGK1 are stable reference genes suitable for normalising expression for genes of interest during in vivo morula to expanded blastocyst development of horse embryos.