251 FACTORS INFLUENCING THE SUCCESS OF EQUINE INTRACYTOPLASMIC SPERM INJECTION IN A CLINICAL PROGRAM

2016 ◽  
Vol 28 (2) ◽  
pp. 258
Author(s):  
K. Hinrichs ◽  
Y. H. Choi

Intracytoplasmic sperm injection (ICSI) is currently being used clinically in horses, but little information is available on factors affecting its success. We have conducted research trials and evaluated data from our clinical ICSI caseload, currently over 450 cases per year, to provide information in this area. In the following summaries, blastocyst data are reported per injected oocyte; clinical data are from 2013 and 2014. Clinically, when immature follicles were aspirated, the number of follicles aspirated per mare decreased significantly with mare age, from 16.2 at age 12–15 to 8.9 at age 24–25; however, maturation and blastocyst rates of recovered oocytes did not differ significantly. Immature oocytes shipped to the laboratory by referring veterinarians yielded a significantly higher blastocyst rate than that for immature oocytes aspirated at the laboratory (27 v. 21%, respectively). Shipped oocytes recovered from stimulated preovulatory follicles yielded a higher blastocyst rate per oocyte than did shipped immature oocytes (39 v. 27%, respectively), but provided fewer mature oocytes per aspiration (1.0 v. 4.5 for immature). From research data, administration of gentamicin and ampicillin to mares before immature oocyte aspiration did not affect blastocyst rate. Holding the aspirate for ~1.5 h at ambient temperature (26 to 33°C) was associated with a blastocyst rate of 32%; however, holding for 2 h at 32°C yielded only 16% blastocysts v. 23% for control. Blastocysts (18%) were obtained from oocytes recovered in the nonbreeding season (December and January). Holding oocytes at room temperature overnight before maturation did not affect blastocyst rate (25 to 34%), nor did inclusion of zinc in the maturation medium (18 to 31%). Diluting and refreezing semen to increase doses available for ICSI did not affect blastocyst rate (23 to 27%); blastocysts (13%) were also obtained after injection of nonmotile sperm. Significant differences in cleavage and blastocyst rates were identified among stallions. For one stallion, use of density gradient plus swim-up before ICSI increased cleavage (49 v. 18%) and blastocyst rates (11 v. 0%) compared to density gradient alone. Blastocyst production was not affected by the amount of glucose added to a human embryo culture medium (0 or 5 mM added glucose on Days 0–5, then 10 or 20 mM added glucose; 31 to 46% blastocysts). Replacement of 10% FBS in embryo culture medium with a mixture of FBS, human serum replacement, and equine follicular fluid lowered blastocyst rate (15 v. 37% for FBS alone). Clinically, embryos were vitrified or were shipped to embryo transfer centers for transfer. There was no significant difference in ongoing pregnancy rate for embryos from shipped immature v. preovulatory oocytes (54 and 69%). For immature oocytes, embryos developing to blastocyst on Day 10 or 11 had a lower ongoing pregnancy rate after transfer (40 and 0%) than did those developing on Days 7 to 9 (51 to 75%). This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by the Clinical Equine ICSI Program, Texas A&M University.

2004 ◽  
Vol 30 (5) ◽  
pp. 372-376 ◽  
Author(s):  
Byung Chul Jee ◽  
Seung Yup Ku ◽  
Chang Suk Suh ◽  
Young Min Choi ◽  
Jung Gu Kim ◽  
...  

2020 ◽  
Vol 32 (2) ◽  
pp. 158
Author(s):  
D. Le Bourhis ◽  
S. Janati Idrissi ◽  
P. Mermillod ◽  
A. Carmen ◽  
P. Salvetti ◽  
...  

Recently, it has been postulated that oviductal extracellular vesicles (oEV) might act as natural nanoshuttles bringing key components (small noncoding RNAs and proteins) of the oviduct into gametes and embryos. Furthermore, co-incubation of frozen-thawed oEV with invitro-produced bovine embryos was reported to increase blastocyst rate and quality (Almiñana et al. 2017 Reproduction 154, 153-168). The objective of this study was to determine the dose-dependent effect of oEV supplementation of embryo culture medium on the invitro development and cryotolerance of embryos. Briefly, oEV were isolated by ultracentrifugation from a pool of oviductal fluids (8 cows/sample) collected at the slaughterhouse at the post-ovulatory stage and ipsilateral to ovulation and stored at −80°C until used. Slaughterhouse-derived bovine oocytes were invitro matured and fertilised with frozen-thawed semen from one bull (4 replicates; 194 presumptive zygotes per group), according to our standard procedures. After IVF, groups of presumptive zygotes (n=20/drop) were cultured under humidified air with 5% CO2, 5% O2 at 38.8°C for 7 days in 30µL of synthetic oviductal fluid-bovine serum albumin supplemented with oEV at different protein concentrations: 0.5, 0.05, or 0.005mgmL−1 and without (control). Cleavage rates were evaluated on Day 2 and blastocyst rates were assessed on Days 6 and 7 (IVF as Day 0). At Day 7, expanded grade 1 blastocysts were evaluated (International Embryo Technology Society classification) and embryos at the expanded grade 1 blastocyst stage were slow frozen in 1.5M ethylene glycol + 0.1M sucrose and stored in liquid nitrogen. For cryotolerance evaluation, embryos were thawed and cultured for 48h in synthetic oviductal fluid-bovine serum albumin + 1% estrous cow serum. Hatching rates were assessed at 48h post-thawing. Data were analysed by a logistic regression mixed model (SAS, SAS Institute Inc.; Glimmix procedure) followed by post-hoc Tukey for multiple comparisons. Differences were considered significant at P<0.05. No differences were observed among the different oEV concentrations tested for cleavage and Day 6 blastocysts. A tendency (P=0.0535) was observed for Day 7 blastocyst rates (19.1±2.8, 29.4±3.3, 16.0±2.6, and 20.6±2.9 for 0.5, 0.05, 0.005mgmL−1, and control, respectively) in favour of the 0.05mgmL−1 group. However, a significant difference (P<0.0288) for Day 7 grade 1 expanded blastocyst rates in favour of the 0.05mgmL−1 group was observed (5.2±1.6, 12.9±2.4, 3.1±1.2, and 9.8±2.2 for 0.5, 0.05, 0.005mgmL−1, and control, respectively). For cryopreserved embryos, hatching rates of frozen-thawed embryos were not significant among experimental groups (81.6±10.2 (n=19), 89.6±6.6 (n=27), 77.2±12.2 (n=10), and 60.2±13.6 (n=23) for 0.5, 0.05, 0.005mgmL−1, and control, respectively). In conclusion, under our experimental conditions, the supplementation of the embryo culture medium with frozen-thawed post-ovulatory oEV at the protein concentration of 0.05mgmL−1 increased the Day 7 grade 1 expanded blastocyst rate. Moreover, we showed a tendency to improve Day 7 blastocyst rates but with no apparent effects on the cryotolerance of embryos. This work was supported by APIS GENE.


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