In vitro embryo production (IVEP) contributes to the quantitative and qualitative aspects of animal reproduction. Nevertheless, inherent technical factors such as oxidative stress can negatively influence the result and this can impair cell metabolism, thus decreasing the rates of in vitro development, and necessitating the supplementation of culture medium with antioxidants. In this context, compounds of natural origin with this property have been highlighted because of the positive results obtained at different stages of IVEP. Thus, this review aims to present the results obtained by using natural antioxidants to minimize the effects of oxidative stress on gametes and embryos. A variety of natural isolated substances and mixtures (essential oils and extracts) have been studied for supplementation of IVEP media, at stages of in vitro maturation, sperm capacitation, in vitro fertilization, and in vitro development of embryos in different mammalian species. Generally, beneficial effects are observed according to the concentration used, thus demonstrating the potential of several natural antioxidants. Therefore, the main challenges in using these compounds as antioxidants during IVEP include proving their efficiency against free radicals and determining the best concentration at each stage. In addition, understanding the mechanisms of action of such antioxidants is crucial to establishing their use in IVEP biotechnology.

Methods for seminal plasma (SP) removal and the selection of collared peccary sperm for fertilization were compared. The experiments evaluated the following: the (I) impact of centrifugation for SP removal before swim-up for sperm selection and (II) a comparison of different Percoll^® gradient densities (PG 45–90% and PG 35–70%). Non-selected sperm served as the control. Sperm quality was assessed based on motility patterns, morphology, membrane functional integrity, viability, reactive oxygen species (ROS), glutathione (GSH), and DNA integrity. Subsequently, the most successful group in the previous experiment and washing by centrifugation (WC) were compared for motility patterns and fertilization using pig oocytes. Swim-up decreased motility and enhanced ROS compared to the control. Centrifugation before swim-up harmed integrity and viability compared to the control. PG 45–90% (96.8 vs. 69.7 vs. 40.7 µm/s) allowed for a better velocity average pathway (VAP), a better velocity straight line, and better linearity (LIN) than those of the control and PG 35–70% (88.4 vs. 56.0 vs. 27.3 µm/s). Thus, PG 45–90% was used for fertilization. PG 45–90% obtained a higher VAP, a higher amplitude of the lateral head, straightness, and higher LIN than those of the control and WC. Cleavage (25.2–26.3%) and morula (8.1–10.5%) rates did not differ between the groups. Therefore, PG 45–90% and WC were efficient in isolating collared peccary sperm capable of fertilizing pig oocytes.

The work aimed (Experiment I) to compare commercial representations of porcine follicle stimulating hormone (FSH, Pluset® vs. Folltropin®) in concentration (10 ?g/mL) and time (24 h) standard (more used in protocols of in vitro maturation, IVM); (Experiment II) to evaluate the best incubation time (6 h vs. 16 h vs. 24 h) and, (Experiment III) analyze varying concentrations (1.0 ?g/mL vs. 2.5 ?g/mL vs. 10.0 ?g/mL) of representations of FSH on the IVM of bovine oocytes. Thus, oocytes were recovered and submitted to IVM under appropriate conditions. After the IVM, oocytes were evaluated for expansion of cumulus cells (CCs), presence of the first polar body (1PB) and metaphase plate (MII). All the data were analyzed by the Fisher exact test (P < 0.05). Initially, in Experiment I, no difference (P > 0.05) was observed in maturation rates of the oocytes incubated with FSH Pluset® or Folltropin®, assessed by expansion of CCs (97.6% vs. 94.3%), presence of 1PB (76.6% vs. 69.4%) and MII (70.0% vs. 68.6%). In Experiment II, when the incubation time with FSH was evaluated, both Pluset® as Folltropin® showed lower rate of expansion of CCs when they were present only in the first 6 h of IVM. As for presence of 1PB, differences were observed in relation to Pluset® while Folltropin® showed similar results in all incubation times. Regarding the MII, no difference was observed between the incubation times with FSH Pluset® and Folltropin®. In Experiment III, no difference was observed in the expansion of CCs, presence of 1PB and MII for concentrations evaluated FSH Pluset® and Folltropin®. Therefore, the FSH Pluset® and Folltropin® have the same efficiency in IVM of bovine oocytes. Regarding the incubation time, it is recommended to maintain FSH (Pluset® or Folltropin®) throughout the period of IVM, since there was no difference in the results of presence of MII. Furthermore, the concentration of 1.0 ?g/mL of FSH Pluset® and Folltropin® is as effective as 10 ?g/mL and can therefore be used for IVM of oocytes.

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 10⁶ spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.