In Vitro Culture of Embryos from LOPU-Derived Goat Oocytes.

A high oocyte quality is the prerequisite for in vitro embryo production. Goat cumulus-oocyte complexes (COC) are mainly collected from slaughterhouse ovaries or by laparoscopic ovum pickup (LOPU) from live animals. Several features can influence the availability of good quality oocytes recovered by the LOPU technique. Interestingly, slaughterhouse and LOPU oocytes have different in vitro maturation kinetics and requirements, and thus, the IVP system must be adapted regarding the oocyte origin. Overall, the use of undefined media in the different steps makes interpretation of results more difficult, hampers their reproducibility, and introduces a sanitary risk. Thus, there is an effort worldwide to use simpler conditions for goat IVP. Although the success of IVP rates is relatively high, in vitro embryos differ from in vivo-derived ones in many aspects, resulting in lower viability. Therefore, strategies to improve in vitro embryo quality are crucial, such as the use of oviductal epithelium cells for coculture. Here we describe the main steps and culture media which can be utilized to produce embryos in vitro from LOPU or slaughterhouse oocytes in goats.

Dose and administration protocol for FSH used for ovarian stimulation affect gene expression in sheep cumulus-oocyte complexes.

The present study evaluated the effect of four ovarian stimulation protocols on the follicular population and molecular status of cumulus-oocyte complexes (COCs). Twelve Santa Inês ewes (in a cross-over design) received 80 or 120mg FSH alone in a multiple-dose (MD80 and MD120) regimen or in combination with 300IU equine chorionic gonadotrophin (eCG) in a one-shot (OS80 and OS120) protocol. The follicular population, COC recovery rate, mean COCs per ewe and the rate of brilliant Cresyl blue-positive (BCB+) COCs were similar among treatments (P>0.05). The expression of markers of oocyte competence (ZAR1, zygote arrest 1; MATER, maternal antigen that embryo requires; GDF9, growth differentiation factor 9; BMP15, bone morphogenetic protein 15; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2 associated X protein) and the steroidogenic pathway (ERα, oestrogen receptor α; LHr, LH receptor; FSHr, FSH receptor; STAR, steroidogenic acute regulatory protein) was affected by stimulation. Specifically, the expression of markers of the steroidogenic pathway was reduced with increasing FSH dose in the OS protocol. FSH at a dose of 80mg reduced the expression of FSHr and ERα in the OS versus MD protocol. Conversely, in MD protocol, only LHr was affected by increasing FSH dose. In conclusion, 80mg FSH in the MD or OS protocol was sufficient to promote the development of multiple follicles and obtain fully grown (BCB+) oocytes. The MD protocol may be more appropriate for the production of better-quality oocytes.

Nonsurgical embryo recovery and transfer in sheep and goats.

The embryo transfer techniques used in small ruminants worldwide are based in surgical procedures. These actions are performed under general anesthesia which needs a combination of animal fasting and drugs for secure animal handling and surgery manipulations. Therefore, it involves risks to animal health and life. The major limiting sequels are adhesions formed by the abdominal surgery, in the ovaries, uterus, or between them. These occurrences can both compromise uterus accessing and oocyte capture and are responsible for decreasing success and limiting successive embryo collections. In contrast, nonsurgical embryo procedures can be performed in a relatively simplified way. Nonsurgical embryo recovery does not need animal prolonged starvation, drug retention is minimized, and donors can stay in a standing position. After the end of embryo recovery, donors are promptly restored to their routine housing and feeding. Furthermore, this technique does not need incisions and, therefore, can be used repetitively in superovulated or nonsuperovulated goats and sheep for embryo recovery-a similar procedure done in cattle. In Brazil, promising results are reported using nonsurgical embryo transfer in recipient goats, and studies are currently evaluating similar procedures in sheep. Therefore, this review aimed to present the current panorama of nonsurgical embryo transfer in sheep and goats.

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up-derived oocytes have different kinetics and requirements regarding maturation media.

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.