The aim of this work was to establish an in vitro culture approach using bovine oviducal fluid (OF) to improve embryo quality and to provide an in vitro system to study oviduct function. Bovine oviducts ipsilateral to ovulation were collected at the slaughterhouse, 1 to 4 days after ovulation. The OF was collected by flushing the oviducts with 1 mL of Charles Rosenkrans 1 medium (CR1). Samples from 21 oviducts were pooled and proteins were concentrated using centrifugal filter devices. Aliquots of 3 different protein concentrations, determined by Bradford assay, were prepared and stored at –20°C. Abattoir-retrieved cumulus–oocyte complexes were used for standard in vitro maturation (IVM) and IVF (Day 0). On Day 1, presumptive zygotes (n = 1498) were randomly allocated to 4 different culture groups and cultured up to Day 9. The presumptive zygotes of the control group (n = 364) were cultured in CR1 with 5% oestrous cow serum (OCS) supplemented with 1 mg mL−1 hyaluronan. In the experimental groups, OCS was replaced by OF, resulting in 3 groups with final protein concentrations of 0.1 mg mL−1 (n = 380), 0.5 mg mL−1 (n = 380) or 1 mg mL−1 (n = 374). Cleavage rate was recorded on Day 2 and blastocyst yield on Days 7, 8, and 9 after fertilization. On Day 7, blastocysts were removed and either stained (Hoechst 33342) for cell number or subjected to a slow freezing protocol using 1.5 M ethylene glycol. After thawing, the re-expansion and hatching rate of blastocysts were determined at 24, 48 and 72 h. Eight replicates were carried out and data were analysed by ANOVA. Cleavage rate increased with increasing protein concentration (0.1 mg mL−1: 80.9 ± 4.2%; P > 0.05; 0.5 mg mL−1: 83.4 ± 2.5%; P < 0.1) and was significantly higher in the 1 mg mL−1 group (84.5 ± 4.4%; P < 0.05) compared with the control group (79.7 ± 3.4%). The cumulative blastocyst rate on Day 9 was significantly lower (P < 0.05) in all experimental groups (0.1 mg mL−1: 15.8 ± 8.9%; 0.5 mg mL−1: 18.7 ± 12.0%; 1 mg mL−1: 17.0 ± 11.2%) compared with the control group (34.1 ± 5.4%). The total number of cells was not affected by OF (P > 0.05). There was no significant difference (P > 0.05) in the post-thaw re-expansion rate between the experimental groups (0.1 mg mL−1: n = 26 thawed blastocysts; 0.5 mg mL−1: n = 27; 1 mg mL−1: n = 23) and the control group (n = 58). The post-thaw hatching rate was significantly higher at 24 and 72 h, respectively, in the 0.5 mg mL−1 group (44.4% and 74.1%; P < 0.05) and the 1 mg mL−1 group (47.8%; P < 0.05; and 82.6%; P < 0.01) compared with the control group (18.9% and 44.8%). The replacement of serum with OF during in vitro culture of bovine embryos had a stage specific effect, resulting in higher cleavage rates but lower blastocyst rates. To address this issue, OF will be collected at different stages and applied in the matching in vitro culture phases in future studies. Interestingly, the post-thaw hatching rate was up to twice as high in the experimental groups, indicating better quality of those embryos developing to blastocyst stage.
On their long path through the female reproductive tract to the fertilization site, spermatozoa are exposed to diverse influences and hazards of the cervical, uterine, and oviducal environment that naturally select viable sperms for the following fertilization. Consequently, this results in a reduction from several billions of sperms in the ejaculate to a functional sperm reservoir within the range of 102 in the isthmus of the Fallopian tube. A technique to deposit spermatozoa directly into the ampulla, thus bypassing most of the reproductive tract, enables a rigorous reduction in number of sperms deposited. Furthermore, it provides a direct assessment of sperm fertility. The aim of our study was to establish an endoscopy-assisted intratubal insemination technique using different sperm dosages, fresh or cryopreserved, to determine adequate conditions for optimal fertilization. Eighteen Simmental heifers were inseminated with fresh semen, and 9 heifers were inseminated with frozen semen using this novel technique. The heifers were synchronized using a modified Ovsynch protocol, and insemination was conducted 18 to 20 h after the second gonadotropin-releasing hormone application. Insemination of heifers was performed under epidural anaesthesia. A tubing system bearing the endoscope and an insemination device was introduced through the vaginal wall into the peritoneal cavity. The insemination device consisted of a tube connected to a curved glass capillary tube loaded with semen. After a visual examination of the ovaries for the presence of an ovulatory Graafian follicle, the capillary tube was inserted directly via the infundibulum into the ipsilateral ampulla and the semen dose was deposited. The entire procedure took ~10 min. Two days later the oviduct was flushed by the same technique. A tubing system connected to a metal catheter served for flushing the embryos and unfertilized oocytes from the oviduct into the uterine horn. Afterward, embryos and oocytes were collected by flushing the uterine horn using an embryo flushing catheter and an embryo filter (EmCon). Embryos were stained using a Hoechst dye to visualise the numbers of attached spermatozoa to the zonae pellucidae. From 18 inseminations with fresh semen doses of 7 to 28 million sperms, 7 embryos at the 2- to 8-cell stage were found. Two of these embryos had more than 10 accessory sperms (AS), 3 had 3 to 6 AS, and 2 were without AS. From 9 inseminations with frozen semen doses containing 1.5 million sperms, we obtained 2 embryos, one at the 4-cell stage without AS and one at the 8-cell stage with 5 AS. Additionally, 3 unfertilized oocytes were collected. In conclusion, these preliminary results demonstrate a promising technique for intratubal AI, which has to be further optimized by studying numbers and treatment of spermatozoa and time of insemination.
Superovulation is a routine procedure to stimulate growth and ovulation of multiple follicles. However, the hormonal changes in the reproductive tract after superovulation treatment affect the environment and subsequently the early embryo development. The aim of the study was to examine the effect of superovulation pretreatment on embryo development and gene expression of IVM/IVF derived embryos subsequently cultured in vivo. The cumulus‐oocyte complexes derived from slaughterhouse ovaries were in vitro matured and fertilized. The denuded presumptive zygotes were cultured in CR1 medium with 5% oestrous cow serum. A total of 788 cleaved embryos at Day 2 were transferred by transvaginal endoscopy into the oviduct of synchronized and superovulated heifers (superstimulated group, SS) and 784 cleaved embryos were transferred into the ipsilateral oviduct of single ovulated synchronized heifers (single ovulation group, SO). In total, 10 Simmental heifers were used for in vivo culture in a crossover design. The in vivo culture was repeated once at an interval of at least 6 weeks in the same animal. At Day 7, embryos were recovered by combined flushing of the oviducts by endoscopy and the adjacent part of the uterine horns by conventional procedure. The numbers of recovered blastocysts were recorded and the embryos were cultured for the following 48 h to determine the blastocyst rate at Days 8 and 9. Simultaneously, 410 cleaved embryos were cultured in vitro for 9 days (control group, C). Triplicate pools of 10 blastocysts recovered at Day 7 from each treatment group were used for RNA isolation. Real-time PCR using sequence specific primers was performed in StepOnePlus™ real time PCR system (Applied Biosystem, Foster City, CA, USA). A comparative threshold cycle method was used to quantify expression levels of the candidate genes compared to the internal control GAPDH gene. The number of recovered embryos after in vivo culture was significantly lower in the SS group compared with the SO group (66.9 v. 79.5%, respectively; P < 0.05). The blastocyst rates at Days 7, 8, and 9 in the SS, SO, and C groups were not significantly different (31.9, 43.3, and 47.1% v. 35.2, 48.5, and 53.5% v. 37.8, 50, and 56.1%, respectively). Molecular analysis of selected genes playing important roles during pre-implantation development revealed significantly lower expression levels of IL6, IL18, and ABCC2 between both experimental in vivo culture groups and the C-group. The IL18 was also significantly down-regulated in the SS-group compared to the SO-group. The transcription factor NFκB was found to be down-regulated in the SS-group compared to the SO and C groups (P < 0.05). In conclusion, we showed that the superovulation pretreatment did not affect blastocyst yield during the culture period but seemed to influence the expression of developmentally important genes in the resulting embryos.
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