Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures.

Cryopreservation and transplantation of ovarian tissue are proposed methods for the restoration of endocrine function and reproductive potential. Therefore, this study aimed to evaluate the effects of vitrification and xenotransplantation on follicle viability, activation, stromal cell integrity, vascularization, and micronuclei formation. Bovine fetal ovaries were fragmented and assigned to the following groups: Fresh control (FC), ovarian fragments immediately fixed; Vitrified control (VC), ovarian fragments vitrified; Vitrified xenotransplanted (VX), ovarian fragments vitrified and xenotransplanted; and Fresh xenotransplanted (FX), ovarian fragments xenotransplanted. Ovarian fragments were grafted in female BALB/c mice and recovered after 14 days. Follicular viability was preserved (P > 0.05) in VC group. The rate of developing follicles was greater (P < 0.05) in the FX group compared to other groups. Follicular density was higher (P < 0.05) in the VC group than the FC, VX, and FX groups. A decrease (P < 0.05) of stromal cell density was recorded after vitrification (VC vs. FX). Blood vessel density decreased in VC, VX, and FX groups compared with the FC group, and blood vessel density was correlated with follicular viability (positively; P = 0.07) and developing follicles (negatively; P < 0.001). Both vitrification and xenotransplantation groups (VC, VX, and FX) had a greater (P < 0.05) number of cells with one MN compared to the FC group. In summary, our findings showed that both vitrification and xenotransplantation modified blood vessel, follicular and stromal cell densities, follicular viability and activation, and micronuclei formation in ovarian tissue.

Embryo production by intracytoplasmic injection of sperm retrieved from neonatal testicular tissue of Agu pigs after cryopreservation and grafting into nude mice.

The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0 = xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1 ± 0.1%, mean ± SEM for four mice), the proportion reached 36.9 ± 16.7% (n = 4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation.

On-farm lambing outcomes after transfer of vitrified and slow frozen embryos.

The aim of the present study was to determine the most suitable embryonic stage and embryo freezing technique for commercial implementation of frozen embryo trading by small-scale sheep producers. There was a 2 × 2 factorial design utilized for conducting the study consisting of two embryo stages (2-8 cells or morula/blastocyst) and two cryopreservation protocols (vitrification or slow-freezing). For the in vivo produced embryos, there were treatments of crossbred donor ewes to induce superovulation. Embryos were recovered surgically on either Day 2 or 5.5 after estrous onset. The embryos were cryopreserved using either a vitrification or slow-freezing method before there was transfer to recipients. Ovarian response, embryo survival and lambing outcomes were analyzed. There were no differences in number of recovered and fertilized embryos at the two embryonic developmental stages. There were no effects of embryonic stages and cryopreservation methods on pregnancy rate, twinning rate, fetal birth weights and lamb weight at 1 month of age. When there was use of vitrified embryos for transfers, there was a greater lamb weight at 2 months of age (8.38 ± 0.20 compared with 7.78 ± 0.21 kg; P = 0.044) than when there was transfer of embryos cryopreserved using slow freezing procedures. Considering economic and practical benefits to small-scale sheep farms, morula/blastocyst stage-embryo collection and transfer into the uterus is more efficacious than transferring 2-8 cells embryos into the oviduct. Results of this study may contribute to the genetic improvement in the flocks of small-scale sheep producers.

Persian onager (Equus hemionus onager) endometrial explant cryopreservation and in vitro culture.

Assisted reproduction of endangered equids, such as Persian onagers (Equus hemionus onager), is vital for species conservation. Little is known about Persian onager reproductive functions, including functions of the uterine endometrium. Recently, successful cryopreservation of the domestic mare endometrium was reported, but there is no information on cryo-sensitivity or in vitro culture of endometrial tissues of any non-domestic equid. In the present study, endometrial explants from Persian onagers were cryopreserved and cultured in vitro for 5 days. There was no difference between endometrial explants when 10% and 20% dimethyl sulfoxide (DMSO) was used for cryopreservation. Cell viability and structural integrity were comparable to fresh tissue. Abundance of estrogen receptor-α (ESR1) and progesterone receptor (PGR) mRNA transcript in endometrial explants was less in most treatment groups compared to the fresh tissue control. There was variation in E-cadherin mRNA abundance in endometrial explants among treatment groups with some treatment groups having a lesser abundance compared to the control group. The abundance of Ki67 mRNA transcript of endometrial explants was not different among treatment groups compared to the control group. Results indicate that DMSO is a suitable cryoprotectant for the Persian onager endometrium, and in vitro culture in a liquid-gas interface can maintain Persian onager endometrial explants for as long as 5 days. Findings allow for a greater understanding of reproductive mechanisms in vitro for this endangered species and other domestic equids including donkeys.

Cold case: Small animal gametes cryobanking.

Germplasm preservation of animals, whether they are valuable domestic breeds or rare species, is the main goal of gamete cryobanking. Dogs and cats act as models for this purpose thanks to the wide availability of biological material which can be employed to experiment protocols that can then be applied to wild animals. This review is focused on spermatozoa, oocytes and gonadal tissues cryobanking in small domestic animals, which is still an unsolved case. Like in a courtroom, evidences of cryoinjuries affecting cellular structures will be presented, penalties as loss of functionality due to cellular alterations will be described, and appeal as strategies to protect gametes from damages or rescue their functionality will be discussed. Differences and similarities between single cell or tissue cryopreservation will be highlighted, together with the rationale for the choice of one type of preservation or another and the fundamental principles which they are based on. The deep analysis of different aspects that still hamper the success of cryopreservation in small animals can help clarify where research is most needed. Therefore, as in a cold case, investigation should remain open in order to hopefully find the solution and make these procedures more and more efficient in the future.

Heterotopic ovarian allotransplantation in goats: Preantral follicle viability and tissue remodeling.

An appropriate implantation site favors angiogenesis and avoids ovarian tissue damage after tissue grafting. The objective of this study was to evaluate the effects of intramuscular (IM) and subcutaneous (SC) sites for ovarian grafts in goats by evaluating follicular morphology and activation, preantral follicle and stromal cell densities, tissue DNA fragmentation, collagen types I and III depositions, and graft revascularizations. Ovarian cortical tissue was transplanted in IM or SC sites and recovered 7 or 15 days post-transplantation. There was a greater percentage of developing follicles and lesser follicular and stromal cell densities in all grafted tissues as compared to ovarian tissues of the control group. The stromal cell density and percentage of normal follicles were positively associated. At 15 days post-transplantation, tissues at the SC and IM sites had similar amounts of DNA fragmentation and type III collagen content. In contrast, tissues at the SC, as compared with IM site, had greater abundances of collagen type I. Furthermore, there was a positive association between collagen type I and percentage of morphologically normal follicles post-transplantation. In addition to a marked decrease in follicular density 15 days post-transplantation in ovarian grafts at the SC and IM sites, low percentages of normal follicles and follicular activation were observed similarly in both transplantation sites. There were also positive associations of stromal cell density and abundance of type I collagen fibers with the percentage of intact follicles in grafted ovarian tissues.

Vitrification leads to transcriptomic modifications of mice ovaries that do not affect folliculogenesis progression.

Ovarian tissue cryopreservation is emerging as a promising alternative for fertility preservation of cancer survivors. To date, more than a hundred couples have successfully had babies using this procedure, although it is still considered experimental and demands further investigation. In this work, we evaluated the effects of vitrification, warming and autotransplantation procedures on the morphology and gene expression of murine ovaries. Ovaries were removed from adult female C57BL6 mice (n = 15), vitrified, warmed and autotransplanted (vitrified group), additionally, ovaries were autotransplanted without vitrification (control group, n = 15). After twenty days, grafted ovaries were harvested and used for histological and ultrastructural analysis, germinal vesicle (GV) oocyte collection, RNA sequencing, and Transmission Electron Microscopy (TEM). All classes of follicles and GV were observed in both control and vitrified/warmed transplanted ovaries, and the numbers of primordial, antral and atretic follicles were not different (p > 0.05). Using RNA-seq, we detected 16,602 vs 13,527 expressed genes in vitrified and control ovaries, respectively; and 623 significantly dysregulated genes (fold change >1.5; 332 up-regulated and 291 down-regulated). Cellular membranes, cytoskeletons, and extracellular matrices were found as the main functions of the differentially expressed genes. Moreover, vitrified samples also presented ultrastructural alterations in the cytoskeleton, cell junctions, and endoplasmic reticulum. Taken together, this work showed for the first time that ovarian cells might trigger a compensatory gene regulation mechanism to maintain cellular structure and folliculogenesis progression after vitrification and autotransplantation.

Resveratrol treatment during maturation enhances developmental competence of oocytes after prolonged ovary storage at 4 °C in the domestic cat model.

Resveratrol (Resv; 3,4,5-trihydroxy-trans-stilbene) is a phytoalexin with antioxidant activity that modulates redox homeostasis in oocytes and improves in vitro embryo production. Cold storage of cat ovaries for a period longer than 24 h alters oxidative status of oocytes after in vitro maturation and reduces their developmental competence. The aim of this study was to evaluate the effect of resveratrol supplementation to the maturation medium on embryo development of oocytes after storage of domestic cat ovaries at 4 °C for 24 h or 48 h. Cumulus-oocyte complexes (COCs) were recovered from ovaries of domestic queens and cultured in maturation medium supplemented with (+) or without (-) 5 µM resveratrol for 24 h. COCs collected from fresh ovaries were matured in vitro (IVM) in standard conditions as control. After IVM, oocytes were in vitro fertilized (IVF) and presumptive zygotes cultured for 7 days. Oocyte nuclear maturation, reactive oxygen species (ROS) and glutathione (GSH) levels as well as cleavage, blastocyst formation and blastocyst cell number were determined. There were no differences in the maturation rates of oocytes between the control and stored groups, irrespective of resveratrol supplementation. Resveratrol treatment during IVM significantly increased the level of GSH and reduced the level of ROS of oocytes recovered from ovaries stored for 48 h as compared to the non-treated group (48 h-). The rate of blastocyst formation from oocytes recovered from ovaries after 48 h storage that underwent IVM with resveratrol was higher (P < 0.05) than that of oocytes matured without resveratrol and similar to that of control oocytes. Resveratrol treatment increased (P < 0.05) cell number in blastocysts from 24 h + and 48 h + groups as compared to their respective counterparts. In conclusion, our results demonstrated that resveratrol supplementation during IVM can reverse the adverse effect of oxidative stress on oocytes, and enhances embryo development after ovary storage at 4 °C for 48 h. These results may provide a basis for improving culture conditions and extend the possibility of storage of cat ovaries for more than 24 h thus ensuring successful in vitro embryo production.

Angiogenesis and follicular development in ovarian tissue of cattle following vitrification and post-warming culture on chicken chorioallantoic membrane.

The purpose of this study was to assess the viability and growth of follicles in ovarian tissues of cattle vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryovial and cryotop). Cortical slices (1-2 mm3) from cattle ovaries (n = 5) were assigned to one of the 14 treatment groups. Cortical slices were vitrified in a TCM199 medium supplemented with ethylene glycol, DMSO, calf serum and either 0.5 M sucrose or trehalose, in cryovials or on cryotops. After warming, cortical slices were either fixed immediately for histology or grafted on a chorioallantoic membrane (CAM) of 10-day old chick embryos. Angiogenesis in ovarian tissues was determined. Viable and atretic preantral (primordial, primary and secondary) follicle densities were examined histologically. There was angiogenesis (chicken) in cortical slices grafted on the CAM by day 5 of culture, however, there was no difference for blood vessel densities when there was use of non-permeating cryoprotectants or cryodevices. Total, viable and atretic follicle densities did not differ (P > 0.05) with use of non-permeating cryoprotectants or cryodevices. The proportion of viable follicles was greater (P < 0.001) in fresh-control than CAM culture-control or vitrification groups. The inclusion of sucrose in the vitrification solution resulted in a larger number of atretic follicles than in the fresh-control group (P < 0.05). In summary, sucrose and trehalose, and cryotop and cryovial were equally suitable for vitrification of ovarian tissues of cattle. Vitrification of ovarian tissues of cattle with subsequent use of CAM culture adversely affected follicular development.

Effect of cryopreservation on the ability of granulosa cells to support in vitro development of oocytes derived from porcine early antral follicles.

Granulosa cells (GCs) contribute to oocyte development. The present study addressed the effect of cryopreservation on the ability of GCs to support oocyte development. GCs were collected from antral follicles. Oocyte granulosa cell complexes (OGCs) derived from early antral follicles were cultured with additional fresh-GCs or frozen-thawed-GCs for 14 days, and the developmental ability and characteristics of the oocytes grown in vitro were examined. Furthermore, fresh- or frozen-thawed-GCs were cultured for two days, and the effects of cryopreservation on the characteristics of GCs were examined. The developmental ability of blastocysts and the acetylation levels of H4K12 in oocytes grown in vitro did not significantly differ among the three culture conditions: OGCs cultured with additional fresh-GCs, frozen-thawed-GCs, or without additional GCs. Although both fresh- and frozen-thawed-GCs exhibited increased ATP content compared with that in oocytes developed without additional GCs, only fresh-GCs showed significantly increased lipid content in oocytes grown in vitro. ATP content, reactive oxygen content, mitochondrial membrane potential, and mitochondrial DNA copy number were greater in cultured frozen-thawed-GCs compared with fresh-GCs. In contrast, lipid content of cultured frozen-thawed-GCs was lower than that of fresh-GCs. Both fresh- and frozen-GCs support oocyte growth, but cryopreservation changes the properties of GCs in a manner that affects the energy status of oocytes grown in vitro.

The cryoprotective effect of Ficoll 70 on the post-warming survival and quality of Cryotop-vitrified donkey embryos.

Many domestic donkey breeds are at risk of extinction, there is a critical urgency for genome resource banking. In the present study, we examined whether the use of Ficoll 70 added to the vitrification medium containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose improves the cryotolerance of donkey in vivo derived embryos. Day 7-8, grade 1-2 donkey embryos were measured and morphologically evaluated and then vitrified-warmed using the Cryotop technique. Before vitrification, embryos were randomly distributed into two groups: (i) VS1 (n = 14): vitrified using 15% EG + 15% DMSO + 0.5 M sucrose; and (ii) VS2 (n = 10): vitrified in the same medium supplemented also with 18% of Ficoll 70. After 24 h of warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). Post-warming survival was numerically higher but not significantly different (P > 0.05) when embryos were vitrified in VS2 (70%) compared to VS1 (57.1%). Embryo rupture was only observed in the VS1 group (21.4%, 3/14). Higher embryo diameter was observed in all groups after 24 h culture (P < 0.05). No significant differences (P > 0.05) were observed among treatments in terms of percentages of cell death. These results demonstrate that the addition of Ficoll 70 to the vitrification medium is not a pre-requisite for successful vitrification of donkey embryos. However, its addition seems to enhance some of the post-warming embryo quality characteristics. Since no statistically significant evidence was found, further studies should be conducted in order to confirm our findings.

Effect of cryopreservation on sperm DNA fragmentation and apoptosis rates in the testicular tissue of domestic cats.

The objectives of the present study were to evaluate the damage caused by cryopreservation on sperm DNA and estimate the percentage of cell apoptosis in tissue after thawing. Testicles of cats were sectioned into of 0.3 cm3 and 0.5 cm3 fragments and evaluated for DNA damage using acridine orange and semi-quantitatively through histo-morphological and immunohistochemical methods (caspase-3). Other fragments were placed in cryotubes with diluent containing either 3% glycerol or 3% propanediol, and were cryopreserved. Evaluation using acridine orange indicated there was a difference with use of propanediol and glycerol on DNA damage in 0.5 cm3fragments, with the latter being more effective than the former for cryopreservation. Results from histomorphological evaluations indicated there was a greater cell integrity among germ cells that were not cryopreserved, based on criteria assessed (detachment of cells from basal membrane, retraction of seminiferous tubule epithelium, visibility of the spermatogonia nucleoli and nuclear spermatogonia condensation), for both sizes of fragments. The values for these variables decreased after cryopreservation, with there being no differences as a result of size of fragment stored or between cryoprotectants used (P > 0.05). The staining for caspase-3 differed for the cytoplasm, nuclei and germ cells. Assessment of these staining patterns indicated the fresh fragments had an amount of cell damage and there was a similar amount of damage detected in cryopreserved fragments. This finding indicated that there was considerable efficacy in preserving the tissue fragments with use of the freezing protocols that were evaluated in this study.

The effect of oleic and linoleic acid addition to the culture media on bovine embryonic development following vitrification.

In this study, the effects of oleic (18:1 cis-9-octadecenoic acid) and linoleic (18:2 (n-6), 9,12-octadecadienoic acid) acids added to the embryo culture media for bovine embryonic development after vitrification were investigated in cattle. Following maturation and fertilization, the oocytes were placed in Charles Rosencrans (CR1aa) culture drops containing 10, 100, 500, and 1000 µM of oleic or linoleic acids. On day 7 or 8 of the culture, the blastocysts and expanded blastocysts were vitrified and warmed to evaluate the viability and development. High doses of oleic acid (1000 µM) in the culture media increased the viability of embryos after vitrification. Similarly, linoleic acid at 1000 µM increased the viability compared to the other linoleic acid doses. It was observed that the addition of essential fatty acids improved the development of embryos. Increasing the concentration of linoleic and oleic acid concentrations in the media proportionally advanced the embryonic development and hatching capability after vitrification/ /warming. Specifically, the addition of high doses of oleic acid had dramatic effects on the embryonic development after vitrification/warming probably due to the increased lipid storage. In conclusion, the present results suggest that the ratio of unsaturated fatty acids in the culture media affects significantly the embryonic development in vitro.

Natural antioxidants in the vitrification solution improve the ovine ovarian tissue preservation.

The present study evaluated the effect of the addition of antioxidants anethole (AN) and robinin (RO) in the vitrification solution, and the in vitro incubation (IVI) medium of ovine ovarian tissue. Ovarian fragments were vitrified without antioxidant (VWA) or with different concentrations of AN (30, 300 and 2000 µg/mL) or RO (0.125, 0.25 and 0.50 mg/mL), followed by IVI (24 h). Histological analyses showed that the percentage of morphologically normal preantral follicles (MNPF) in AN 2000 did not differ from RO 0.125 or fresh ovarian tissue (CTR). Subsequently, ovarian fragments were vitrified in the presence of AN 2000 and RO 0.125 followed by IVI without or with (AN 2000+ and RO 0.125+) the same antioxidants. The follicular activation in all treatments was significantly increased as compared to the CTR. The stroma cell density (SCD) in all the vitrified fragments was significantly lower than the CTR. However, in the AN 2000 and RO 0.125 this parameter was significantly higher when compared to the VWA. The reactive oxygen species (ROS) in the ovarian cortex of the AN 2000 or AN 2000+ were significantly reduced in comparison with the CTR while the intracellular ROS levels of AN 2000 and CTR were similar. The total antioxidant capacity (TAC) in RO 0.125 was significantly higher than that of VWA, AN 2000 and AN 2000+. According to the results, the use of antioxidants (AN or RO) only in the vitrification solution of ovine ovarian tissue is recommended, due to their better preservation of the SCD. Moreover, AN 2000 best maintains the follicular morphology, while RO 0.125 has a high TAC.

The recipient's Day after ovulation and the number of corpora lutea influence the likelihood of pregnancy in mares following transfer of ICSI frozen embryos.

Asynchronous embryo transfer is an excellent tool to investigate how subtle differences in the uterine environment affect embryo development and survival. Progesterone secreted from the corpus luteum following ovulation is one of the main factors responsible for establishing endometrial receptivity for the pre-implantation embryo via complex alterations in the expression of genes involved in the secretion of the histotroph. The objective of this retrospective study was to determine whether the recipient's Day after ovulation and the number of CL at ET influence the pregnancy rates of IVP horse embryos. The study included 650 heterologous frozen ICSI horse embryo transfer cycles and evaluated the pregnancy and ongoing pregnancy rate. The ongoing pregnancy was significantly lower in recipient mares with ET performed 5 and 6 days after ovulation (47.4% and 37.5%, respectively) than in recipients with ET 4 days after ovulation (67.3%). Furthermore, Day 5 recipient mares (Day 0 = Day of ovulation) with 2 corpora lutea (CL) at the time of ET had lower ongoing pregnancy rate (36.1%) than Day 5 recipient mares with 1 CL (51.9%). In contrast, the presence of 2 CL was associated with a higher ongoing pregnancy rate (75.8%) in recipient mares with a less advanced uterine stage at the time of ET (Day 3 and 4 after ovulation), compared to recipients with only 1 CL at ET (62.7%). In conclusion, both the number of days after ovulation and the number of CL recorded in the recipient mare at ET influenced the ongoing clinical pregnancy rate. This study highlights the importance of establishing exactly when progesterone rises above a threshold (relative to the Day of ovulation) when trying to determine the optimal window for transferring an IVF/IVP embryo.

Effect of cryoprotectant type and concentration on the vitrification of collared peccary (Pecari tajacu) ovarian tissue.

The aim of the present study was to establish a protocol for solid surface vitrification of peccary ovarian tissue by using different cryoprotectants. Ovarian pairs from five adult females were fragmented and two fragments (fresh control group) were immediately subjected to morphological evaluation using classical histology, transmission electron microscopy, and viability analysis using fluorescent probes. The remaining fragments (n = 18) were vitrified using a solid surface method with different concentrations (3 or 6 M) of ethylene glycol (EG), dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). After 2 weeks, samples were re-warmed and evaluated. A decrease in the percentage of morphologically normal preantral follicles (PFs) was verified for all the groups in comparison to the fresh control (92.0 ± 2.8%); however, if only the primordial follicles are considered, the most effective preservation (P < 0.05) was achieved with the use of EG at 3 M (74.2±7.3%) or DMSO at 6 M (75.0 ± 4.2%). Ultrastructural analysis indicated there were well-preserved PFs in all the groups evaluated, having well-defined membranes, a few vacuoles, and organelles that were uniformly distributed throughout the cytoplasm, mainly round and elongated mitochondria in close association with lipid droplets. Viability was preserved (P < 0.05) with the use of EG at 3 (97%) or 6 (97%) M, DMSO at 3 (100%), and DMF at 6 (97%) M. Solid surface vitrification, therefore, is an effective method for conservation of peccary female germplasm, especially with the use of EG at 3 M, which was highly effective for preservation of both the morphology and viability of PFs.

Effect of macromolecule supplement on nuclear and cytoplasmic maturation, cryosurvival and in vitro embryo development of dromedary camel oocytes.

The current evaluation of oocyte vitro maturation (IVM) media has progressed toward more defined conditions in human and livestock. In this study, the replacement of fetal calf serum (FCS) with bovine serum albumin (BSA) and polyvinyl alcohol (PVA) was evaluated during IVM in dromedary camel. Nuclear maturation rates in presence of FCS and PVA were comparable (81.6 ±â€¯1 and 75.5 ±â€¯5%, respectively). BSA, whether used alone or in combination with FCS, significantly reduced nuclear maturation (51.6 ±â€¯3.9 and 54.6 ±â€¯1.1%, respectively), compared to FCS and PVA. BSA also increased the rates of chromosome aberrations compared to FCS and PVA (25.7 ±â€¯7.4, 8.8 ±â€¯2.3 and 6.0 ±â€¯2.0%, respectively). IVM macromolecule differentially affected morphological aspects of cumulus expansion and FCS promoted the highest dissociation of cumulus cells, compared to all the other groups. FCS significantly increased mean lipid intensity of oocytes compared to BSA, FCS-BSA and PVA which could explain the lower cryo-survival of oocytes matured in presence of FCS compared to BSA and PVA (56.1 ±â€¯5.2, 91.0 ±â€¯19.5, and 87.8 ±â€¯6.7%, respectively). Mitochondrial activity was not affected by macromolecules, but oocytes cultured with PVA had the best redox status, compared to other IVM groups. Cleavage was not affected by IVM macromolecule, but FCS promoted significantly higher rate of morula development (51.6 ±â€¯5.2 vs. 33.6 ±â€¯2.9% for PVA) and blastocyst development (36.8 ±â€¯1.4 vs. 20.5 ±â€¯2.0% for BSA). Although adding FCS during IVM supported highest hatching rate of the resulting blastocysts, differential cell number showed no long lasting effect of IVM macromolecules on blastocyst quality. Obtained results suggest the possibility to switch from undefined to more defined IVM systems for efficient in vitro maturation and subsequent vitrification of dromedary camel oocytes. Keywords: camel, oocyte maturation, protein supplement, cryosurvival.

Innovative, Simple Models for Teaching Neuroanatomy Using the Elnady Technique.

Plastination is a valuable tool for the teaching of neuroanatomy. However, the high cost of the process and the complexity of sheet plastination for brain slices remains a challenge. This article describes an innovative, simple, and inexpensive method, called the Elnady Technique, to develop brain slices of various domestic animals. The slices are either enveloped in lamination sheets using an electric iron, or enveloped in transparent plastic using an impulse sealer. This fast, effortless process results in realistic, durable, odorless, soft, flexible slices. The models provide accurate three-dimensional (3D) reference guides for demonstration of neuroanatomical structures that show soft tissue contrast between the gray and white matter. This makes them invaluable for interpretation of clinical imaging modalities, such as computed tomography (CT) and magnetic resonance imaging (MRI). These ethically sourced models can provide a replacement for the killing of animals for practical classes.

Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue.

Preservation of cellular integrity and its mechanisms after ovarian tissue cryopreservation (OTC) and in vitro culture (IVC) procedures are crucial aspects for the success of preservation and recovery of female fertility. This study aimed to evaluate the effects of two cryopreservation methods (slow-freezing, SF, and vitrification, VIT) on the equine ovarian tissue after 1, 3, and 7 days of IVC by assessing: (i) preantral follicle morphology and distribution of follicle classes; (ii) protein expression of markers of cell proliferation for EGFR and Ki-67; (iii) markers of apoptosis for Bax and Bcl-2; and (iv) DNA fragmentation. Percentages of normal primordial follicles were similar (P > 0.05) among SF-control, VIT-control, and fresh control groups. After 7 days of culture, VIT-IVC7 had a greater (P < 0.05) total percentage of normal preantral follicles when compared with SF-IVC7, but both had a lower (P < 0.05) percentage than fresh IVC7 group. Prior to and after 7 days of culture, expression of EGFR and Ki-67 were similar (P > 0.05) among fresh, SF, and VIT groups. After 7 days of culture, VIT had higher (P < 0.05) Bax expression than the fresh and SF tissues, but Bcl-2 was similar (P > 0.05) among groups. Prior to IVC, TUNEL signals were similar (P > 0.05) among groups; however, VIT-IVC7 had greater (P < 0.05) TUNEL signals when compared with the fresh IVC7 group. In conclusion, findings demonstrated: (i) similar efficiency between SF and VIT compared with fresh control to preserve morphologically normal follicles; and (ii) similar tissue functionality and cell proliferation capability after equine OTC by either SF and VIT methods following IVC for 7 days. The results herein presented shed light on equine fertility preservation programs using OTC techniques.

An optimized controlled rate slow cooling protocol for bovine ovarian tissue cryopreservation by means of X-ray computed tomography.

Cryopreservation and subsequent transplantation of ovarian tissue is the only option to preserve fertility in certain patients facing gonadotoxic treatment. So far, cryopreservation of ovarian tissue has been carried out mostly by a controlled rate slow cooling process, typically known as slow freezing. Even though there are still some concerns about the iatrogenic damage on the follicle population, this technique has been used in the more than 100 live births reported to date. It is well known that the control of the cryoprotectant loading in the tissue is crucial to in a cryopreservation procedure. We have used the technology of X-ray computed tomography to assess the concentration and distribution of dimethyl sulfoxide (one of the cryoprotectants most used in fertility preservation) inside pieces of bovine ovarian tissue after its cryopreservation. The low voltage used in our device (75 kV) and the high electronic density of this cryoprotectant makes the X-ray attenuation proportional to its concentration. By assessing and comparing the permeation and homogeneity of the cryoprotectant inside ovarian tissue fragments subjected to a controlled rate slow cooling process, we have characterized the effect of variations in the main parameters involved in the process, with the goal of achieving an optimized protocol with higher permeation of the cryoprotectant in the tissue. The most promissory results were obtained by increasing the initial concentration of dimethyl sulfoxide in the vehicle solution from 10 to 20%v/v.