Successful equine in vitro embryo production by ICSI - effect of season, mares' age, breed, and phase of the estrous cycle on embryo production.

This retrospective study aimed at identifying factors that contribute to the success of equine in vitro embryo production by intracytoplasmic sperm injection (ICSI). A total of 7993 ovum pick-up (OPU) sessions were performed, totaling 2540 donor mares and semen from 396 stallions. Oocytes were aspirated at multiple sites in Brazil and were sent to the laboratory, within 6 h from OPU, in pre-maturation medium where they were in vitro matured (IVM) followed by ICSI and in vitro embryo culture for 7-8 days. The number of recovered oocytes, matured oocytes, cleaved embryos and blastocysts were used to explore the effect of age and breed of the donor mare, time of year in which the mare was aspirated and phase of the estrous cycle on the day of follicular aspiration. Mares between 6 and 15 years old were superior to other age groups in most parameters evaluated, including the average number of blastocysts per OPU. The impact of age was similar when evaluated within two breeds, American Quarter Horse (AQHA) and Warmblood mares. We observed that breed (AQHA, Warmblood, Crioulo, Lusitano and Mangalarga) had an important effect on most of the parameter evaluated, including number of oocytes recovered, blastocysts produced per OPU, and blastocyst rates. The overall impact of season was less pronounced than age and breed, with the only statistically significant difference being a higher rate of oocyte maturation during the summer season. Finally, most of the parameters evaluated were superior in follicular phase mares, with or without dominant follicle than luteal phase mares. In conclusion, this retrospective study revealed that breed, age, season and stage of estrous at the time of OPU are all important parameters for the success of equine embryo production by ICSI. This technology enables producing embryos all-year-round from mares of different breeds and ages from OPU-derived oocytes collected at multiple sites.

Effect of inducing accessory corpus luteum formation with gonadotropin-releasing hormone or human chorionic gonadotropin on the day of embryo transfer on fertility of recipient dairy heifers and lactating cows.

The objective was to determine the effect of inducing an accessory corpus luteum (CL) using GnRH or human chorionic gonadotropin (hCG) on the day of in vitro-produced (IVP) embryo transfer (ET) on pregnancy per ET (P/ET) and calving/ET in dairy heifers and lactating cows. Dairy heifers (11-15 mo of age; n = 1,547) and lactating cows (n = 1,480) detected in estrus by tail chalk (d 0) were used as recipients. Before ET, the presence of a CL was evaluated by transrectal palpation from d 6 to 9 of the estrous cycle. Animals with a CL were randomly assigned to receive 1 of 3 treatments immediately before ET: control (no treatment; n = 1,009), GnRH (86 µg of GnRH; n = 1,085) and hCG (2,500 IU; n = 1,069). Embryos were implanted in the uterine horn ipsilateral to the ovary with a CL (fresh IVP embryos, n = 2,544; vitrified IVP embryos n = 545; slow-freezing IVP embryos, n = 74). Pregnancy diagnosis was performed on d 37 ± 3 of gestation by transrectal palpation. Pregnancy loss data and calving records were collected from the dairy farm management software. Treatment did not affect P/ET, calving/ET, or pregnancy loss either overall or within parity. When treatments inducing CL formation were combined (GnRH + hCG), heifers tended to have greater P/ET than controls (67.7 vs. 63.5%, respectively). Yet, calving/ET were similar. Response variables were also analyzed within embryo type and parity. For heifers receiving stage 6 (blastocyst) fresh IVP embryos, hCG had greater P/ET than controls (74.5 vs. 51.1%, respectively). In addition, GnRH tended to have greater P/ET than controls (67.8 vs. 51.2%, respectively). However, calving/ET in heifers receiving blastocyst fresh IVP embryos was similar among treatments. When only stage 7 (expanded blastocyst) fresh IVP embryos were considered, primiparous GnRH cows had greater P/ET (59.3 vs. 47.1%) and calving/ET (48.6 vs. 38.1%) than hCG. Moreover, hCG showed decreased calving/ET compared with controls in primiparous cows transferred with expanded blastocyst fresh IVP embryos. In summary, the effects of hCG or GnRH at ET on P/ET and calving/ET were inconsistent according to different embryo characteristics (e.g., embryo stage) and parity of recipients. Furthermore, treatment did not improve the overall fertility outcomes for recipient animals receiving IVP embryos.

Single embryo and oocyte lipid fingerprinting by mass spectrometry.

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species.

Mass spectrometry fingerprinting of media used for in vitro production of bovine embryos.

Using the bovine species as a biological model, direct infusion chip-based nano-electrospray ionization mass spectrometry (nano-ESI-MS) fingerprinting in the positive ion mode is used to obtain fast chemical profiles of media used for in vitro production of bovine embryos. Nano-ESI-MS fingerprinting is useful for characterization and routine quality control requiring no sample pre-separation, being able to differentiate four different media (IVM, IVF, SOF and HSOF) via principal component analysis (PCA). For media stored at +4 degrees C for up to 45 days, no significant (p>0.05) variation was observed in cleavage and blastocyst rate development, as well as in the nano-ESI-MS chemical profiles. For media exposed to a heat shock (60 degrees C for 3 h), no significant decrease (p>0.05) in embryo development rates was observed, but nano-ESI-MS profiles were quite distant from fresh control media in the PCA. For frozen media (-70 degrees C for 2 months), again no significant variation (p>0.05) in embryo development was noticed, but nano-ESI-MS profiles from all media were significantly affected. These results indicate that nano-ESI(+)-MS fingerprinting was able to characterize different media based on their specific chemical profile. The technique seems therefore applicable as a routine quality control assay, detecting, for example, compositional changes after temperature variations that may affect post-transfer embryo viability.