In vitro embryo production in small ruminants: what is still missing?

In vitro embryo production (IVEP) is an extremely important tool for genetic improvement in livestock and it is the biotechnology that has grown the most recently. However, multiple ovulation followed by embryo transfer is still considered the leading biotechnology for embryo production in small ruminants. This review aimed to identify what is still missing for more efficient diffusion of IVEP in small ruminants, going through the IVEP steps and highlighting the main factors affecting the outcomes. Oocyte quality is essential for the success of IVEP and an aspect to be considered in small ruminants is their reproductive seasonality and strategies to mitigate the effect of season. The logistics for oocyte collection from live females is more complex than in cattle, and tools to simplify this collection system and/or to promote an alternative way of recovering oocytes may be an important point in this scenario. The heterogeneity of oocytes collected from growing follicles in live females or from ovaries collected from abattoirs remains a challenge, and there is a demand to standardize/homogenize the hormonal stimulatory protocols and IVM protocols for each source of oocytes. The use of sexed semen is technically possible, however the low market demand associated with the high costs of the sexing process prevents the routine use of this technique, but its higher availability is an important aspect aiming for greater dissemination of IVEP. New noninvasive approaches for embryo selection are key factors since the selection for transfer or cryopreservation is another difficulty faced among laboratories. Embryo selection is based on morphological traits, although these are not necessarily reliable in predicting pregnancy. Several issues described in this review must be considered by researchers in other to promote the diffusion of IVEP in small ruminants.

Luteinizing hormone secretion, ovulatory capacity, and oocyte quality in peripubertal Gir heifers.

Induction of puberty in cattle breeds that attain puberty in later stages, such as Gir, allows the earlier beginning of reproductive life and it might increase oocyte quality. Here, the ovulatory capacity of prepuberal Gir heifers was studied and its relationship to follicular growth, luteinizing hormone (LH) secretion and oocyte quality was evaluated. Peripubertal Gir heifers were treated with a progesterone-based protocol and according to ovulatory response were separated into groups: not-ovulated (N-OV) and ovulated (OV). Serial blood samples were taken 24 h after estradiol treatment on day 12 to evaluate LH secretion. Cumulus-oocyte complexes (COCs) were collected using ovum pick-up and assessed for brilliant cresyl blue (BCB) staining rate, IVF-grade oocytes rate, and mean oocyte diameter, in comparison with cow oocytes. Gene expression of developmental competence markers (ZAR1, MATER, and IGF2R) was also analyzed. The largest follicle diameters were similar between N-OV and OV groups on the day of estradiol treatment (d12) and the next day and decreased (P = 0.04) in the N-OV group thereafter. LH pulse secretion was different between groups (N-OV = 3.61 ± 0.34 vs OV = 2.83 ± 0.21 ng/ ml; P = 0.04). COC assessment showed that the number of recovered oocytes, BCB+ rate, IVF-grade oocytes and oocyte size was similar (P > 0.05) among groups, resembling adult cow patterns. ZAR1, MATER and IGF2R gene expression in oocytes were also similar (P > 0.05) in N-OV and OV groups. In conclusion, our results demonstrate a lower LH secretion profile in peripubertal Gir heifers prone to ovulate after induction protocol, and that oocyte quality is not affected on a short-term basis by ovulation itself.

The Simulated Physiological Oocyte Maturation (SPOM) system in domestic animals: A systematic review.

Simulated Physiological Oocyte Maturation (SPOM) mimics in vitro the physiological events of oocyte maturation. The system uses cAMP modulators in two steps (pre IVM and IVM) and has presented promising results that are arousing the curiosity of IVF programs in different animal species, generating several papers, adaptations, and controversies worldwide. This study systematically analyses the data in the literature on the use of SPOM and compares the outcomes to the original paper (Albuz et al. Hum. Rep., 25: 2999-3011 2010), classifying them into success or failure. The PubMed, Scopus, and Google Scholar databases were searched and 22 studies were included, from which data on 26 experiments were extracted and evaluated via descriptive statistical analysis. Only experiments that assessed the blastocyst rate (BR) were considered for the success parameter, i.e. success (increase in BR) or failure (either no difference or a reduction in BR). The experiments applied the SPOM system in the following species: cattle, sheep, goats, mice, mares and cats. Three experiments (3/26) could not be evaluated for success or failure, and of the remaining, 34.7% (8/23) succeeded in improving blastocyst production. More than two-thirds (69.2%, 18/26) of experiments were conducted in cattle; of those, 86.8% (13/15) used TCM-199 as the IVM media, and 22.2% did not use forskolin or IBMX modulators as indicated in the original study. Also, 27.7% (5/18) of the experiments in cattle used the same type and dose of FSH, and 22% (4/18) used the same protein source and concentration as indicated in the original study. All experiments conducted in mice (3) kept the parameters of the original study in terms of forskolin and IBMX doses and BSA and FSH concentrations, however, they removed cilostamide from IVM. Cilostamide was used during IVM in more than half (53.8%) of all experiments, but only in cattle and sheep. Considering oocyte and embryo assessments, six experiments assessed cAMP levels and most (5/6) of these observed an increase: in cattle (2), sheep (2), and mice (1). Ten experiments evaluated the effect of SPOM on nuclear maturation, and in 90% (9/10), the SPOM system was able to arrest meiosis (cattle, sheep and mice). Thirteen experiments evaluated the total cell number (cattle, mice and sheep), and six (6/13) showed an increase. Our findings clearly indicate difficulties in reproducing the SPOM system worldwide, demonstrating that the meiosis arrest is not sufficient to ensure successful SPOM application. They also suggest that the different supplements used in the IVM medium and/or their interaction with modulators for different durations may produce a significant bias that affects experimental success.

Optical imaging of cleavage stage bovine embryos using hyperspectral and confocal approaches reveals metabolic differences between on-time and fast-developing embryos.

The assessment of embryo quality aims to enhance subsequent pregnancy and live birth outcomes. Metabolic analysis of embryos has immense potential in this regard. As a step towards this goal, here we assess the metabolism of bovine embryos using label-free optical imaging. We compared embryos defined as either on-time or fast-developing, as fast dividing embryos are more likely to develop to the blastocyst stage. Specifically, bovine embryos at 48 (Day 2) and 96 (Day 4) hours post fertilization were fixed and separated based on morphological assessment: on-time (Day 2: 2 cell; Day 4: 5-7 cell) or fast-developing (Day 2: 3-7 cell; Day 4: 8-16 cell). Embryos with different developmental rates on Day 2 and Day 4 were correlated with metabolic activity and DNA damage. Confocal microscopy was used to assess metabolic activity by quantification of cellular autofluorescence specific for the endogenous fluorophores NAD(P)H and FAD with a subsequent calculation of the optical redox ratio. Separately, hyperspectral microscopy was employed to assess a broader range of endogenous fluorophores. DNA damage was determined using γH2AX immunohistochemistry. Hyperspectral imaging showed significantly lower abundance of endogenous fluorophores in fast-developing compared to on-time embryos on Day 2, indicating a lower metabolic activity. On Day 4 of development there was no difference in the abundance of FAD between on-time and fast-developing embryos. There was, however, significantly higher levels of NAD(P)H in fast-developing embryos leading to a significantly lower optical redox ratio when compared to on-time embryos. Collectively, these results demonstrate that fast-developing embryos present a 'quiet' metabolic pattern on Day 2 and Day 4 of development, compared to on-time embryos. There was no difference in the level of DNA damage between on-time and fast-developing embryos on either day of development. To our knowledge, this is the first collective use of confocal and hyperspectral imaging in cleavage-stage bovine embryos in the absence of fluorescent tags.

The SPOM-adapted IVM system improves in vitro production of bovine embryos.

This study aimed to test the effects of an IVM SPOM adaptation (SPOM-adapted IVM) on the production, total number of cells (TNC), apoptosis, and cryotolerance (post-warming survival and cytoskeleton actin integrity) of bovine IVP embryos. Two experiments were conducted with two experimental groups based on IVM treatment: A control group (TCM 199 without FCS) and an SPOM-adapted group (TCM 199 with forskolin and IBMX in pre-IVM and IVM with cilostamide). The first experiment evaluated embryo in vitro production, TNC, and apoptosis rate on D9 of development. In the second experiment, embryos were vitrified/warmed at D7 (control fresh and vitrified; SPOM-adapted fresh and vitrified) and assessed regarding post-warming survival rates and cytoskeleton actin integrity. Statistical analysis was performed using GraphPad INSTAT software at a significance level of 5%. An increase (p < 0.05) in blastocyst production was observed in the SPOM-adapted group comparing to the control group. There was no difference (p > 0.05) in the TNC or apoptosis rate between the groups. Regarding cryopreservation, no differences were found (p > 0.05) in actin integrity or post-warming survival rates between the vitrified groups. In both vitrified groups, we observed a significantly lower uninjured pattern of actin integrity compared to the fresh groups (p < 0.05). We conclude that the SPOM-adapted IVM system is beneficial for blastocyst production and does not affect the quality and cryotolerance of the produced embryos.

Breed composition does not influence the performance of Holstein-Gyr crossbred as oocyte donors for OPU/IVP.

Holstein-Gyr crossbred cattle are strategic for dairy systems in tropical countries, since they combine milk yield genetics with adaptability to tropical climate. However, Holstein (Bos taurus) and Gyr (Bos indicus) breeds present remarkable differences regarding reproductive physiology. Brazil stands out as the world's largest user of embryo in vitro production (IVP) in bovine, and the use of this technique is increasing in dairy systems. As Holstein-Gyr crossbreds are important oocyte donors for IVP, the present work aimed at investigating whether increased Gyr or Holstein breed composition influences donor's performance. Sixteen Holstein-Gyr crossbred females presenting increased (HG, 71.4 to 87.5% Holstein; n = 9) or decreased (GH, 40.2 to 46.6% Holstein; n = 7) Holstein composition were submitted to three ovum pick up (OPU) sessions. We observed similar (P = 0.2946) antral follicle count between HG and GH donors (24.8 ± 3.2 vs 29.4 ± 2.8 respectively; mean ± SEM). Groups also display similar morphological oocyte grading (Grade I: 0.1 ± 0.1 vs 0.1 ± 0.1 - P = 0.9680; Grade II: 0.9 ± 0.5 vs 1.9 ± 0.5 - P = 0.1942; Grade III, 4.0 ± 1.2 vs 7.2 ± 1.4 - P = 0.1047, HG vs GH respectively; mean ± SEM). Additionally, the proportion of viable oocyte was similar between HG and GH groups (27.8% vs 31.9%, respectively, P = 0.3500) and oocyte lipid area fraction (6.8% vs 9.5%, respectively; P = 0.1539). Our results indicate that the individual variation has more influence than breed composition of crossbred oocyte donors.