There are few reports of cryopreservation and injuries in Macrobrachium amazonicum embryos. Thus, the aim of this study was to analyze the effects of cryoprotectants agents and cooling on stage VIII of this species. Fertilized eggs from ovigerous females were removed from the incubation chamber, then placed in 10 ml Falcon tubes with a cryoprotectant solution and saline-free calcium solution. Thus, the embryos underwent a cooling curve of 1°C per min until reaching 5°C, and then were stored for 2 h. The tubes containing the embryos were washed to remove the cryoprotectant, acclimated for 5 min and then transferred to 50 ml incubators. At the end of the 24-h period, living embryos from each tube were counted and tabulated. A pool of embryos was fixed with 4% formaldehyde and then subjected to histology using 3-mm thick sections and stained with haematoxylin/eosin. Another pool was used for biometric analysis in which length, width and volume were analyzed. The cryoprotectants agents used were: dimethylsulfoxide (DMSO), methyl alcohol, ethylene glycol at 1, 5 and 10% and sucrose (0.5 M). Variance analysis was performed followed by Tukey's honest significant difference (HSD) test at 5% significance level. DMSO cryoprotectant affected embryo survival the least with rates of 71.8, 36.2 and 0% for concentrations of 1, 5 and 10%, respectively. Ethylene glycol caused 100% mortality at all the concentrations used. It was not possible to observe the interference of cooling and cryoprotectants on embryonic structures in this study.

The aim of this study was to compare the effects of various activating factors on feline oocytes. The study included activation within the ovary (natural), activation during in vitro maturation (spontaneous activation), chemical activation (ionomycin + 6-DMAP), activation by spermatozoa and injection (ICSI) and mechanical activation (sham ICSI). According to our results, parthenogenetic embryos could emerge at every step of in vitro embryo production (IVP) procedures. After oocyte collection, 6% of parthenogenetic embryos were observed, mainly at the 2–4-blastomere stages. After 24 h of in vitro maturation, parthenogenetic activation was observed in 7% of oocytes. Using ionomycin and 6-DMAP to artificially activate oocytes, 53% of cleaved embryos were obtained. The results after ICSI (54% cleaved embryos) were not significantly different from the results in Group III using chemical activation (53% cleaved embryos). But only after ICSI were blastocysts obtained (5/73.7%) as a result of in vitro culture. Moreover, embryos after ICSI were of the best morphological quality with minor levels of fragmentation evident in the embryos. After sham mechanical activation, ‘sham ICSI’, 8% of cleaved embryos were noted. Therefore, it is advised to maintain a negative control in parallel with each step of IVP techniques, to avoid misleading results. Chemical methods for artificial activation of feline oocytes are the most promising for application to the cloning and production of parthenogenetic embryos for experimental studies.

The Deleted in AZoospermia (DAZ) gene family regulates the development, maturation and maintenance of germ cells and spermatogenesis in mammals. The DAZ family consists of two autosomal genes, Boule and Dazl (Daz-like), and the Daz gene on chromosome Y. The aim of this study was to analyze the localization of DAZL and BOULE during testicular ontogeny of the seasonal-breeding Syrian hamster, Mesocricetus auratus. We also evaluated the testicular expression of DAZ family genes under short- or long-photoperiod conditions. In the pre-pubertal and adult testis, DAZL protein was found mainly in spermatogonia. BOULE was found in the spermatogonia from 20 days of age and during the pre-pubertal and adult period it was also detected in spermatocytes and round spermatids. DAZL and BOULE expression in spermatogonia was strictly nuclear only in 20-day-old hamsters. We also detected the novel mRNA and protein expression of BOULE in Leydig cells. In adult hamsters, Dazl expression was increased in regressed testis compared with non-regressed testis and DAZL protein expression was restricted to primary spermatocytes in regressed testis. These results show that DAZL and BOULE are expressed in spermatogonia at early stages in the Syrian hamster, then both proteins translocate to the cytoplasm when meiosis starts. In the adult regressed testis, the absence of DAZL in spermatogonia might be related to the decrease in germ cell number, suggesting that DAZ gene family expression is involved in changes in seminiferous epithelium during photoregression.

The aim of this study was to describe the effect of temperature on the fertilization, early developmental stages, and survival rate of two Neotropical catfishes Pimelodus maculatus and Pseudopimelodus mangurus. After fertilization, the eggs were incubated at 22°C, 26°C, and 30°C, which resulted in fertilization rates of 96.95 ± 1.79%, 98.74 ± 0.76%, and 98.44 ± 0.19% for P. maculatus and 96.10 ± 1.58%, 98.00 ± 0.63%, and 94.60 ± 2.09% for P. mangurus, respectively. For P. maculatus, hatching occurred after 22 h 30 min post-fertilization at 22°C, 16 h 30 min at 26°C, and 11 h 20 min at 30°C, and the hatching rates were 43.87 ± 7,46%, 57.57 ± 17.49%, and 53.63 ± 16.27%, respectively. For P. mangurus, hatching occurred after 28 h 30 min post-fertilization at 22°C and 17 h 30 min at 26°C with respective hatching rates of 45.4 ± 21.02% and 68.1 ± 12.67%. For this species, all embryos incubated at 30°C died before hatching. Additionally, for P. maculatus, the larvae from the lower (22°C) and higher temperatures (30°C) presented increased abnormality rates, as observed in the head, tail and yolk regions. The lowest abnormality rate was detected at 26°C, which was considered the optimal incubation temperature for both species. The developed protocol enables the manipulation of embryonic development, which is important for the application of reproductive biotechniques, including chimerism and chromosome-set manipulation. The data obtained here are also important for the surrogate propagation of this species as P. mangurus was recently categorized as an endangered fish species.

The objective of the study was to investigate the effect of l-ergothioneine (l-erg) (5 mM or 10 mM) supplementation in maturation medium on the developmental potential and OCTN1-dependant l-erg-mediated (10 mM) change in mRNA abundance of apoptotic (Bcl2, Bax, Casp3 and PCNA) and antioxidant (GPx, SOD1, SOD2 and CAT) genes in sheep oocytes and developmental stages of embryos produced in vitro. Oocytes matured with l-erg (10 mM) reduced their embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH in matured oocytes that in turn improved developmental potential, resulting in significantly (P < 0.05) higher percentages of cleavage (53.72% vs 38.86, 46.56%), morulae (34.36% vs 20.62, 25.84%) and blastocysts (14.83% vs 6.98, 9.26%) compared with other lower concentrations (0 mM and 5 mM) of l-erg without change in maturation rate. l-Erg (10 mM) treatment did not influence the mRNA abundance of the majority of apoptotic and antioxidant genes studied in the matured oocytes and developmental stages of embryo. A gene expression study found that the SLC22A4 gene that encodes OCTN1, an integral membrane protein and specific transporter of l-erg was not expressed in oocytes and developmental stages of embryos. Therefore it was concluded from the study that although there was improvement in the developmental potential of sheep embryos by l-erg supplementation in maturation medium, there was no change in the expression of the majority of the genes studied due to the absence of the SLC22A4 gene in oocytes and embryos that encode OCTN1, which is responsible for transportation of l-erg across the membrane to alter gene expression.

The aim of the current study was to investigate the effect of caffeine supplementation during in vitro maturation (IVM) for different maturation times on the developmental potential of canine oocytes recovered from ovariohysterectomized bitches. The recovered cumulus–oocytes complexes were in vitro matured for 72 h. Here, 10 mM caffeine was added to the maturation medium for different incubation times (caffeine from 0–72 h maturation, caffeine for the first 24 h of maturation only, caffeine addition from 24 to 48 h maturation time, caffeine addition from 48 to 72 h maturation or in caffeine-free medium, control group). The matured oocytes were in vitro fertilized using frozen–thawed spermatozoa. The presumptive zygotes were in vitro cultured in synthetic oviductal fluid medium for 5 days. The results showed that both maturation and fertilization rates were significantly higher (P ˂ 0.05) using caffeine-treated medium for the first 24 h of maturation compared with the control and other two groups of caffeine treatment (from 24 to 48 h and from 48 to 72 h), whereas use of caffeine-treated medium for a 0–72 h incubation time did not affect these rates (P > 0.05). Interestingly, the matured oocytes in caffeine-supplemented medium for the first 24 h or from 0–72 h showed a significant (P ˂ 0.05) increase in the total number of cleaved embryos compared with the control group. In conclusion, supplementation of the maturation medium with 10 mM caffeine for the first 24 h of maturation or during the whole maturation time (0–72 h) improved nuclear maturation and subsequent embryo development preimplantation following in vitro fertilization.

This study aimed to understand how germ cell development occurs in females of Devario aequipinnatus, by morphologically describing oogenesis and the reproductive phases. Sexually mature females of D. aequipinnatus (n = 70) were obtained from commercial fisheries and delivered to the Laboratório de Ictiologia Neotropical, UNESP, Ilha Solteira, SP, Brazil. The ovaries were removed, fragmented and fixed following the usual techniques for light microscopy. The stages of ovarian development in D. aequipinnatus begin with the oogonia, which proliferate into new cells or differentiate into prophasic oocytes that, at the end of this process, form the ovarian follicle and end folliculogenesis. In the previtellogenic stage, the oocytes were characterized mainly by the gradual loss of basophilia and an increase in oocyte diameter. Vitellogenesis was marked mainly by the incorporation of yolk granules. Mature oocytes were defined by their migration from the nucleus to the micropyle. Postovulatory follicles and atresic oocytes were also observed. The reproductive phases were classified as: immature, early and final developing, spawning capable, regressing and regenerating. Therefore, the development of an understanding of cell modifications that occurs up to oogenesis is a basic step that is essential for the description of the reproductive biology of D. aequipinnatus, given the lack of information about the reproductive aspects of this species.

To investigate the effects of physiologically relevant heat shock during oocyte maturation, buffalo cumulus–oocyte complexes (COCs) were cultured at 38.5°C (control) or were exposed to 39.5°C (T1) or 40.5°C (T2) for the first 6 h of in vitro maturation (IVM), followed by 38.5°C through the next 18 h/IVM and early embryonic development up to the blastocyst stage. Gene expression analysis was performed on selected target genes (HSF-1, HSF-2, HSP-70, HSP-90, BAX, p53, SOD1, COX1, MAPK14) in denuded oocytes and their isolated cumulus cells resulting from control COCs as well as from COCs exposed to a temperature of 39.5°C (T1). The results indicated that heat shock significantly (P < 0.01) decreased the maturation rate in T1 and T2 cells compared with the control. After in vitro fertilization (IVF), cleavage rate was lower (P < 0.01) for oocytes exposed to heat stress, and the percentage of oocytes arrested at the 2- or 4-cell stage was higher (P < 0.01) than that of the control. The percentage of oocytes that developed to the 8-cell, 16-cell or blastocyst stage was lower (P < 0.01) in both T1 and T2 groups compared with the control group. mRNA expression levels for the studied genes were decreased (P < 0.05) in treated oocytes (T1) except for HSP-90 and HSF-1, which were increased. In cumulus cells isolated from COCs (T1), the expression for the target genes was upregulated except for BAX, which was downregulated. The results of this study demonstrated that exposure of buffalo oocytes to elevated temperatures for 6 h severely compromised their developmental competence and gene expression.