Search

This pioneering study aimed to evaluate the cryopreservation of semen from P. falkneri (n = 4) and P. motoro (n = 4), maintained ex situ at the Sao Paulo Aquarium, Brazil. For this purpose, the animals were physically restrained, biometric data of the disc and clasper were obtained, and semen was collected through manual massage. Total motility and progressive motility parameters were evaluated using Computer-Assisted Sperm Analysis (CASA) with IVOS II equipment and Animal Breeders II software. The semen extenders INRA 96 and OptiXcell were used to assess their efficacy in sperm cryopreservation. INRA required the addition of 5% dimethyl sulfoxide (DMSO) as a cryoprotectant. The results indicated that there was no difference in semen motility values before and after freezing with INRA + DMSO (p = 0.6226). On the other hand, samples cryopreserved with OptiXcell showed a difference in semen motility post-thaw (p = 0.0156). These findings contribute to a broader study on optimizing cryopreservation protocols to ensure long-term viability and fertility of semen, enhancing genetic diversity and supporting wild population restoration. A multidisciplinary approach integrating reproductive biology, ecology, physiology, and assisted reproduction technologies, aligned with the One Conservation concept, is essential for advancing conservation and management strategies for these threatened species. [ABSTRACT FROM AUTHOR]

Preserving genetic material in cryogenic conditions presents a viable alternative for the protection of species' gene variability. However, there is an enormous need to establish and test cryopreservation protocols that are suitable for each diverse cell type to guarantee technical success in the long run. Considering this, fibroblasts from jaguar ( Panthera onca ), oncilla ( Leopardus tigrinus ), and pampas cat ( Leopardus colocolo ) were subjected to cell characterization and then cryopreservation in different cryoprotectant solutions (2.5%, 10% dimethyl sulfoxide [DMSO] or CryoSOfree™). Further testing was conducted to determine each solution's performance in preserving cell viability. In culture, a growth curve to assess cellular growth potential showed that exponential proliferation lasts for about the first 50 hours of in vitro culturing, declining in pace afterward. L. colocolo and L. tigrinus presented no difference in cell viability while using 2.5% DMSO protocols. P. onca cells did not present difference on viability for both concentrations of DMSO. Protocols using CryoSOfree resulted in a decreased viability of P. onca fibroblasts. Morphological differences between fibroblasts among the species were noted under bright field microscopy and scanning electron microscopy. L. colocolo and P. onca cells are fusiform, and L. tigrinus are spherical. All cells presented cytoplasmic projections. Transmission electron microscopy revealed vacuoles and secretion granules, indicating intense cell activity after thawing. Differences found in the efficiency of cryopreservation protocols according to the type of cryoprotectant indicate that species react differently to freezing and thawing processes. This research evaluates key aspects of in vitro protocols for cryopreservation of wild animals, which need to be optimized to guarantee successful cell culturing. More suitable protocols lead to increased efficiency in establishing fibroblast cryobanks and also facilitating the use of wild cats' cells in cloning techniques, contributing directly to preserving wild fauna.

This study aimed to evaluate the effect of sodium caseinate added into freezing extender on the sperm parameters of cryopreserved bull semen and in vitro and in vivo fertility. One ejaculate of 30 bulls was used and processed using Botu-Bov (Botupharma, Botucatu, Brazil) with the addition of 20% egg yolk (EY) or 15% egg yolk with 2% sodium caseinate (EY + SC), subsequently submitted to freezing. Semen from both groups were evaluated immediately after thawing (T0) and after thermic stress at 37 °C for 90 min (T90), for sperm kinetics, by CASA method, and plasma membrane integrity (PMI), superoxide (O 2 - ) concentration and high mitochondrial potential (HMP) by flow cytometry. In vitro fertilization (IVF) was performed to assess embryo cleavage rate on day 3, and blastocyst rate on day 8. The in vivo fertility test was performed using fixed-time artificial insemination (FTAI). In sperm evaluation, trajectory velocity, linear velocity, curvilinear velocity, and lateral head movement were higher (P < 0.05) in EY + SC at T0. At T90, while rectilinearity and linearity did not differ between EY and EY + SC (P > 0.05), the other parameters evaluated were higher in EY + SC. Similarly, the integrity of the plasma and acrosomal membranes (iPAM) was higher (P < 0.05) at T90 in EY + SC, but did not differ (P > 0.05) between the groups at T0. For O 2 - and HMP, the values were lower (P < 0.05) in EY + SC group in both moments; furthermore, EY + SC showed higher cleavage and blastocyst rates in IVF. Likewise, pregnancy rates by FTAI were higher (P < 0.05) in the EY + SC group. In conclusion, the addition of sodium caseinate into freezing extender improves sperm parameters of frozen-thawed bull semen and fertility rates on during in vitro and in vivo tests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sperm cryopreservation offers many benefits to wild felids conservation programs. However, the implementation of these programs is limited by the different responses of each species to the cryopreservation protocols and extenders used, requiring the formulation of species-specific protocols. For this purpose, semen samples from 6 margays (Leopardus wiedii) were submitted to 2 cryopreservation protocols: 1) manual freezing (cooling rate of - 0.33 °C/min at 5 °C/180 min and freezing rate with two steps - 9 °C/min for 2 min and -19.1 °C/min for 2 min) and 2) automatic freezing machine (cooling rate of - 0.25 °C/min at 5 °C/120 min and freezing rate with one step -20 °C/min for 8.3 min) using 2 commercial extenders, an egg yolk-based (Test Yolk Buffer; TYB) and an egg yolk-free extender (AndroMed; MED). Post-thawed sperm quality was assessed at 3 time points (immediately after thawing and 1 and 2 h post-thawed) by sperm motility index (SMI), plasma membrane and acrosomal integrity, and mitochondrial membrane potential (MMP). Regarding SMI, TYB yielded superior results (29.4 ± 3.5%) compared to MED (11.2 ± 2.8%; p < 0.002) immediately after thawing until 2 h after thawing (TYB 3.9 ± 1.7% and MED 0.0 ± 0.0%; p < 0.05). Furthermore, the automated freezing method provided higher motility compared to the manual freezing procedure immediately post-thaw (25.08 ± 3.66% and 15.78 ± 3.29%, respectively) and 1 h post-thaw (13.71 ± 2.56% and 6.03 ± 1.97%, respectively; p < 0.05). The percentage of intact acrosomes and plasma membranes and the percentage of sperm with high MMP were superior for TYB when compared to MED regardless of cryopreservation protocol (p < 0.05). Conversely, the interaction between cryopreservation protocols and extenders was observed for MMP where TYB exhibits better results compared to MED (p < 0.05) in both procedures, but it was higher in automated procedures. For MED, no changes were found in MMP between procedures. Considering only TYB, samples showed higher MMP when submitted to an automated procedure (p < 0.05). In conclusion, the slow cooling rates with shorter time of exposure to glycerol contributed to minimize cryodamage in the Margays' sperm. Moreover, results indicated that association between TYB and automatic freezing machine ensured the minimal quality of spermatozoa after thawing required for further use in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI)., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human embryo cryopreservation techniques enable the storage of surplus embryos created during assisted reproduction procedures; however, the existence of these same surplus embryos has sparked further debate. What can be their fate once they are no longer desired by their parents or if the parents are deceased? Thus, the level of interest in the cryopreservation of oocytes has increased, as has the necessity for further scientific study. This study had the objective of reporting 10 years of experience of freezing and thawing human oocytes from patients who did not wish to freeze embryos. A total of 159 cycles using frozen–thawed oocytes were performed (mean age 33.7 years). Survival and fertilization rates were 57.4% and 67.2%, respectively. Cleavage rate was 88.4% and the pregnancy rate was 37.7%. Clinical pregnancy was observed in 43 cycles (27.0%) with 14.5% of transferred embryos implanted. These pregnancies delivered 19 boys and 23 girls, two pregnancies are ongoing and nine were miscarriages. The average gestational week was 37.6 weeks and birthweight was 2829.2 g. These data suggest that the use of frozen–thawed oocytes in IVF represents a reasonable alternative for those patients not comfortable with the cryopreservation of supernumerary embryos., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

The Brazilian freshwater fish diversity is the richest in the world. Only 0.7% of all Brazilian species have had any aspect of their sperm biology addressed up to this date. The majority of the fish species described in this review migrate during the spawning season (a phenomenon known as piracema). Urbanization, pollution, hydroelectric dams and deforestation are some of the causes of stock depletion or even local extinction of some of these species. The knowledge concerning sperm quality and minimum sperm:egg ratio is important to maximize the use of males without reducing hatching rates. Furthermore, sperm cryopreservation and gene banking can guarantee the conservation of genetic diversity and development of adequate breeding programs of native fish species. In this review, we present and evaluate the existing information on Brazilian fish species that have been subject to sperm quality and cryopreservation studies. The following parameters were evaluated: volume of extractable sperm, sperm motility, sperm concentration, freezing media, freezing methods, and post-thaw sperm quality. Although the existing protocols yield relatively high post-thaw motility and fertilization rates, the use of cryopreserved sperm in routine hatchery production is still limited in Brazil.