Breakthrough Acute HIV Infections among Pre-Exposure Prophylaxis Users with High Adherence: A Narrative Review.

Pre-exposure prophylaxis (PrEP) is a pivotal intervention among HIV prevention strategies. We aimed to narratively revise the topic of HIV acute infection in the setting of PrEP exposure with a focus on diagnostic options, clinical features, and future PrEP perspectives, with a particular focus on users with high adherence to PrEP. We searched the main databases (PubMed, Embase, and Scopus) with the keywords "PrEP" or "Pre-Exposure Prophylaxis" and "HIV" or "PLWH" and "breakthrough" or "acute infection" or "primary infection". We included all randomized clinical trials and non-experimental studies (both case reports and observational studies) ever published. In the present narrative review, we revise the diagnostic challenges related to HIV diagnosis in the setting of PrEP and the clinical characteristics and symptoms of breakthrough infections. We discuss the management of acute HIV infection during PrEP and the new challenges that arise from the use of long-acting drugs for PrEP. Our review underlines that although extremely rare, HIV seroconversions are still possible during PrEP, even in a context of high adherence. Efforts to promptly identify these events must be included in the PrEP follow-up in order to minimize the chance of overlooked HIV breakthrough infections and thus exposure to suboptimal concentrations of antiretrovirals.

Melatonin reduces oxidative stress and improves follicular morphology in feline (Felis catus) vitrified ovarian tissue.

Ovarian tissue vitrification is associated with multiple events that promote accumulation of ROS (reactive oxygen species) which culminate in follicular apoptosis. Thus, this study was aimed at evaluating the role of melatonin in vitrification and culture of feline (Felis catus) ovarian tissue. In phase 1, domestic cat ovaries were fragmented into equal circular pieces of 1.5 mm diameter by 1 mm thickness and divided into four groups (fresh control and 3 treatments). The treatments were exposed to vitrification solutions supplemented with melatonin at 0 M, 10-9 M, and 10-7 M, then vitrified-warmed, histologically evaluated and assayed for ROS. Consequently, phase 2 experiment was designed wherein ovarian fragments were divided into two groups. One group was exposed to vitrification solution without melatonin and the other with 10-7 M melatonin supplementation, then vitrified-warmed and cultured for ten days with fresh ovarian fragments as control prior to assessment for histology, immunohistochemistry (Ki-67, MCM-7 and caspase-3) and ROS. Concentration of ROS was lower (p = 0.0009) in 10-7 M supplemented group in addition to higher proportion of grade 1 follicles. After culture, proportions of intact and activated follicles were higher (p < 0.05) in melatonin supplemented group evidenced by higher expression of Ki-67 and MCM-7. Follicular apoptosis was lower in melatonin supplemented group. In conclusion, melatonin at 10-7 M concentration preserved follicular morphological integrity while reducing ROS concentration in vitrified-warmed feline ovarian tissue. It has also promoted the follicular viability and activation with reduced apoptosis during in vitro culture of vitrified-warmed feline ovarian tissue.

Vitrification of feline ovarian tissue: Comparison of protocols based on equilibration time and temperature.

Global contraction of biodiversity pushed most members of Felidae into threatened or endangered list except the domestic cat (Felis catus) thence preferred as the best model for conservation studies. One of the emerging conservation strategies is vitrification of ovarian tissue which is field-friendly but not yet standardized. Thus, our main goal was to establish a suitable vitrification protocol for feline ovarian tissue in field condition. Feline ovarian tissue fragments were punched with biopsy punch (1.5 mm diameter) and divided into 4 groups. Group 1 was fresh control (Fr), while the other three were exposed to 3 vitrification protocols (VIT_CT, VIT_RT1 and VIT_RT2). VIT_CT involved two step equilibrations in solutions containing dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 10 min each at 4 °C. VIT_RT1 involved three step equilibration in solutions containing DMSO, EG, polyvinylpyrrolidone and sucrose for 14 min in total at room temperature, while in VIT_RT2 all conditions remained the same as in VIT_RT1 except equilibration timing which was reduced by half. After vitrification and warming, fragments were morphologically evaluated and then cultured for six days. Subsequently, follicular morphology, cellular proliferation (expression of Ki-67, MCM-7) and apoptosis (expression of caspase-3) were evaluated, and data obtained were analysed using generalised linear mixed model and chi square tests. Proportions of intact follicles were higher in Fr (P = 0.0001) and VIT_RT2 (P = 0.0383) in comparison to the other protocols both post warming and after the six-day culture. Generally, most follicles remained at primordial state which was confirmed by the low expression of Ki-67, MCM-7 markers. In conclusion, VIT_RT2 protocol, which has lower equilibration time at room temperature has proven superior thus recommended for vitrification of feline ovarian tissue.

Effect of Fixatives and Fixation Period on Morphology and Immunohistochemistry of Feline Ovarian Tissue.

Fixatives and fixation protocol have a profound effect on both the morphology and epitope sensitivity of ovarian tissue, which hampers accurate ovarian tissue evaluation. We aimed to establish the most suitable fixation protocol for feline (Felis catus) ovarian tissue. Fragments (1.5 mm diameter) were punched from 1 mm-thick feline ovarian tissue, divided into three groups then fixed with three different fixatives (Bouin, neutral buffered formalin [NBF] and form acetic acid [new compound fixative formulation for ovarian tissue composed of 5% acetic acid in NBF]) for five fixation periods. Subsequently, fragments were processed and evaluated for the morphology and intensity of immunohistochemical signals against three antigens (Ki-67, MCM-7 and activated caspase-3). Proportions of grade 1 or morphologically intact follicles were significantly lower in NBF when compared with Bouin and form acetic acid fixatives. However, Bouin fixative had the lowest mean DAB intensity (p < 0.05) in all three antigen targets, while NBF had the highest (p < 0.05) in Ki-67 and caspase-3, but in MCM-7, it was no different from form acetic acid. In conclusion, form acetic acid maintained ovarian tissue architecture with excellent follicular morphology in the same manner as Bouin fixative, and it also maintained reasonable DAB signals similar to NBF, thus providing a better alternative for feline ovarian tissue studies.

Different effects of the companion antiretroviral drugs on dolutegravir trough concentrations.

BACKGROUND: Dolutegravir is widely used in different dual and triple antiretroviral regimens. Here, we sought to investigate the effect of the companion antiretroviral drug(s) on dolutegravir plasma trough concentrations in persons with HIV, with a focus on dual regimens. METHODS: Dolutegravir concentrations collected from October 2015 to March 2023 ( n  = 900) were stratified according to the main antiretroviral classes (NRTIs, NNRTIs, protease inhibitors) and according to single drugs. Dolutegravir concentrations measured in persons with HIV concomitantly treated with lamivudine were considered as the reference group. RESULTS: Dolutegravir trough concentrations were significantly higher in persons with HIV given protease inhibitors compared with the reference [1886 (1036-2940) versus 1575 (1026-2226) ng/ml; P  = 0.004]. The highest dolutegravir concentrations were measured in persons with HIV concomitantly treated with unboosted atazanavir [2908 (2130-4135) ng/ml]. Conversely, co-administration of darunavir/ritonavir resulted in significantly lower dolutegravir exposure [909 (496-1397) ng/ml; P  = 0.002 versus reference]. Among NNRTIs, the higher dolutegravir concentrations were measured in presence of rilpivirine [2252 (1489-2686); P  < 0.001 versus reference]. CONCLUSION: Dolutegravir trough concentrations are differently affected by individual antiretroviral drugs, with some drug combinations (i.e. dolutegravir/darunavir/cobicistat, or dolutegravir/rilpivirine) providing significantly higher than expected dolutegravir exposure. Such combinations might be advantageous when there are concerns about dolutegravir plasma exposure or resistance.

Culture of vitrified bovine ovarian tissue on agarose gel inserts maintains follicle integrity.

In brief: Ovarian tissue cryopreservation and culture provide an option for fertility preservation without tissue grafting, but need optimization. This study reveals that vitrified bovine ovarian tissue can be cultured on agarose gel and maintain follicle morphology, low activation, and low apoptosis. Abstract: Ovarian tissue preservation is hitherto a promising fertility insurance option for precious animals. Ovarian tissue vitrification and culture combined approach would eliminate the need of transplanting ovarian tissue to obtain mature oocytes. We aimed at optimizing vitrification and in vitro culture conditions for improved bovine ovarian tissue viability. Ovaries obtained from the slaughterhouse were punched into fragments and divided into three groups. Group 1 (fresh) was divided into two and immediately placed in two-culture systems (culture inserts and agarose inserts). Group 2 was vitrified, warmed, and placed in the two-culture systems, while group 3 was only equilibrated and then placed in the two-culture systems. All cultures were maintained for 6 days and spent media were collected on alternate days for cytokine (interleukin 1ß and interleukin 6) evaluation. Fragments were fixed for morphology assessment and immunohistochemistry. Higher percentages (P < 0.05) of grade 1 (morphologically intact) follicles were observed in fragments on agarose compared to those on culture inserts on days 2 and 4 of the culture. Conversely, we found higher (P < 0.05) shifts of primordial follicles to transitional follicles in fragments on culture inserts vis-à-vis agarose inserts which was consistent with a higher proportion of Ki-67 and MCM-7 and activated caspase-3-positive follicles. In conclusion, in vitro culture of bovine ovarian tissue on agarose inserts maintained follicle morphology, low follicle activation, and low apoptosis compared to culture inserts.

Neuraxial anesthesia for abdominal surgery, beyond the pandemic: a feasibility pilot study of 70 patients in a suburban hospital.

The aim of this study is to establish the feasibility of awake laparotomy under neuraxial anesthesia (NA) in a suburban hospital. A retrospective analysis of the results of a consecutive series of 70 patients undergoing awake abdominal surgery under NA at the Department of Surgery of our Hospital from February 11th, 2020 to October 20th, 2021 was conducted. The series includes 43 cases of urgent surgical care (2020) and 27 cases of elective abdominal surgery on frail patients (2021). Seventeen procedures (24.3%) required sedation to better control patient discomfort. Only in 4/70 (5.7%) cases, conversion to general anesthesia (GA) was necessary. Conversion to GA was not related to American Society of Anesthesiology (ASA) score or operative time. Only one of the four cases requiring conversion to GA was admitted to the Intensive Care Unit (ICU) postoperatively. Fifteen patients (21.4%) required postoperative ICU support. A statistically non-significant association was observed between conversion to GA and postoperative ICU admission. The mortality rate was 8.5% (6 patients). Five out of six deaths occurred while in the ICU. All six were frail patients. None of these deaths was related to a complication of NA. Awake laparotomy under NA has confirmed its feasibility and safety in times of scarcity of resources and therapeutic restrictions, even in the most frail patients. We believe that this approach should be considered as an useful asset, especially for suburban hospitals.

Microenvironment factors promoting the quality of vitrified cat oocytes.

In oocyte cryopreservation programs, vitrification has overthrown conventional slow freezing both in veterinary and human medicine. In animals, its feasibility in field conditions makes it the preferred technique for the safeguard of genetic resources from zoo or wild animals, including threatened felids, for which the domestic cat is an excellent model. However, many cellular injuries, such as cytoskeleton, mitochondria and meiotic spindle alterations, DNA damage, zona pellucida hardening and cumulus cell loss, might occur following vitrification. After warming, although the exact mechanisms are still unclear, degeneration is a frequent outcome for cat vitrified oocytes. For immature (germinal vesicle) gametes, in vitro maturation after warming is a challenge, and cleavage after fertilization barely reaches 15-30%, while for mature (metaphase II) cryopreserved gametes it can get to 30-50%. Anyway, the progression to late embryos stages is often impaired, and improvements are needed. Standard cryopreservation protocol and the use of conventional in vitro culture systems after warming may not be enough for vitrified oocytes to recover and demonstrate their full developmental potential. Physical or chemical factors applied to oocytes undergoing vitrification, as an enrichment to the vitrification step, or to the culture microenvironment, could create more favorable conditions and promote vitrified oocyte survival and development. From the use of three-dimensional culture systems to the regulation of metabolic activities and cellular pathways, this review aims to explore all the possibilities employed so far, including the studies performed by our own lab, and the future perspectives, to present the most effective strategies for cat oocyte vitrification and the best time for their application (i.e., before, during, or after vitrification-warming).

Progress toward species-tailored prematuration approaches in carnivores.

In the past four decades, the bovine model has been highly informative and inspiring to assisted reproductive technologies (ART) in other species. Most of the recent advances in ART have come from studies in cattle, particularly those unveiling the importance of several processes that must be recapitulated in vitro to ensure the proper development of the oocyte. The maintenance of structural and functional communications between the cumulus cells and the oocyte and a well-orchestrated chromatin remodeling with the gradual silencing of transcriptional activity represent essential processes for the progressive acquisition of oocyte developmental competence. These markers are now considered the milestones of physiological approaches to increase the efficiency of reproductive technologies. Different in vitro approaches have been proposed. In particular, the so-called "pre-IVM" or "prematuration" is a culture step performed before in vitro maturation (IVM) to support the completion of the oocyte differentiation process. Although these attempts only partially improved the embryo quality and yield, they currently represent a proof of principle that oocytes retrieved from an ovary or an ovarian batch shouldn't be treated as a whole and that tailored approaches can be developed for culturing competent oocytes in several species, including humans. An advancement in ART's efficiency would be desirable in carnivores, where the success is still limited. Since the progress in reproductive medicine has often come from comparative studies, this review highlights aspects that have been critical in other species and how they may be extended to carnivores.

Epidemiological and Clinical Features of SARS-CoV-2 Variants Circulating between April-December 2021 in Italy.

SARS-CoV-2 is constantly evolving, leading to new variants. We analysed data from 4400 SARS-CoV-2-positive samples in order to pursue epidemiological variant surveillance and to evaluate their impact on public health in Italy in the period of April-December 2021. The main circulating strain (76.2%) was the Delta variant, followed by the Alpha (13.3%), the Omicron (5.3%), and the Gamma variants (2.9%). The B.1.1 lineages, Eta, Beta, Iota, Mu, and Kappa variants, represented around 1% of cases. There were 48.2% of subjects who had not been vaccinated, and they had a lower median age compared to the vaccinated subjects (47 vs. 61 years). An increasing number of infections in the vaccinated subjects were observed over time, with the highest proportion in November (85.2%). The variants correlated with clinical status; the largest proportion of symptomatic patients (59.6%) was observed with the Delta variant, while subjects harbouring the Gamma variant showed the highest proportion of asymptomatic infection (21.6%), albeit also deaths (5.4%). The Omicron variant was only found in the vaccinated subjects, of which 47% had been hospitalised. The diffusivity and pathogenicity associated with the different SARS-CoV-2 variants are likely to have relevant public health implications, both at the national and international levels. Our study provides data on the rapid changes in the epidemiological landscape of the SARS-CoV-2 variants in Italy.

Ultra-Rapid Freezing Preserves Morphofunctional Integrity and Fertilizing Ability of Epididymal Cat Spermatozoa.

Vitrification and ultra-rapid freezing, which are more commonly used for oocytes and embryos, have recently been applied to spermatozoa in an attempt to make semen cryopreservation in field conditions easier compared to conventional freezing. It is well-known that in case of unexpected death of rare and wild animals, preserving epididymal spermatozoa from isolated testicles represents a great chance of salvaging male germplasm for future use in assisted reproductive technologies. The aim of this study was to evaluate the morphofunctional integrity of cat epididymal spermatozoa ultra-rapid frozen in pellets or straws with two different extenders [E1 (Tris buffer with 20% egg yolk and 0.25 M sucrose) or E2 (Ham's F10 with 1% bovine serum albumin and 0.4 M sucrose)] and to test whether spermatozoa preserved by the best combination were able to fertilize oocytes and produce embryos in vitro by intracytoplasmic sperm injection (ICSI) of in vitro matured cat oocytes. The results showed that E1 and E2 in straw or pellet were comparable (at warming, about 30% normal morphology, 45% intact membranes, and 20% intact acrosomes), except for post-warming motility that was better maintained along time by E1 pellet (21.7 ± 7.4% at warming and 3.6 ± 2.9% after 6 h). Such spermatozoa could fertilize conspecific oocytes and support embryonic development (cleavage 35.5%) as well as frozen control spermatozoa (cleavage 54.29%, p = 0.22). In conclusion, cat epididymal spermatozoa better maintained their morphofunctional features after ultra-rapid freezing with E1 and could successfully produce embryos in vitro after ICSI. This underscores their usefulness as cryobanked material for fertility and biodiversity preservation purposes.

Mortality rates among COVID-19 patients hospitalised during the first three waves of the epidemic in Milan, Italy: A prospective observational study.

INTRODUCTION: This paper describes how mortality among hospitalised COVID-19 patients changed during the first three waves of the epidemic in Italy. METHODS: This prospective cohort study used the Kaplan-Meier method to analyse the time-dependent probability of death of all of the patients admitted to a COVID-19 referral centre in Milan, Italy, during the three consecutive periods of: 21 February-31 July 2020 (first wave, W1), 1 August 2020-31 January 2021 (second wave, W2), and 1 February-30 April 2021 (third wave, W3). Cox models were used to examine the association between death and the period of admission after adjusting for age, biological sex, the time from symptom onset to admission, disease severity upon admission, obesity, and the comorbidity burden. RESULTS: Of the 2,023 COVID-19 patients admitted to our hospital during the study period, 553 (27.3%) were admitted during W1, 838 (41.5%) during W2, and 632 (31.2%) during W3. The crude mortality rate during W1, W2 and W3 was respectively 21.3%, 23.7% and 15.8%. After adjusting for potential confounders, hospitalisation during W2 or W3 was independently associated with a significantly lower risk of death than hospitalisation during W1 (adjusted hazard ratios [AHRs]: 0.75, 95% confidence interval [CI] 0.59-0.95, and 0.58, 95% CI 0.44-0.77). Among the patients aged >75 years, there was no significant difference in the probability of death during the three waves (AHRs during W2 and W3 vs W1: 0.93, 95% CI 0.65-1.33, and 0.88, 95% CI 0.59-1.32), whereas those presenting with critical disease during W2 and W3 were at significantly lower risk of dying than those admitted during W1 (AHRs 0.61, 95% CI 0.43-0.88, and 0.44, 95% CI 0.28-0.70). CONCLUSIONS: Hospitalisation during W2 and W3 was associated with a reduced risk of COVID-19 death in comparison with W1, but there was no difference in survival probability in patients aged >75 years.

Freezability of Dog Semen after Collection in Field Conditions and Cooled Transport.

Dog semen freezing is gaining popularity, but it has to be performed in equipped facilities, which can be far from the place where the stud dog lives. The aim of this study was to evaluate whether freezing dog semen after 24 or 48 h of cooled transport to an equipped laboratory was possible when semen collection was performed in the field such as in local breeding kennels. Single ejaculates from different dogs (mixed breeds and ages) were collected. In Experiment I, 10 ejaculates were conventionally frozen using the Uppsala method or frozen after 24 or 48 h of storage in a Styrofoam transport box cooled by icepacks. In Experiment II, 10 ejaculates were used to assess the influence of two extenders (Uppsala chilling extender or freezing extender 1) used for semen dilution during the 24 or 48 h storage. Motility, morphology, membrane, and acrosome integrity were analyzed as well as spermatozoa zona-binding ability. No significant differences were observed among the frozen groups, regardless of freezing time (Experiment I) or extender (Experiment II). Motility at thawing, however, decreased in absolute value at 48 h. Freezing of freshly collected semen is the gold standard, but the results obtained in this study prompt the application of freezing after cooled transport for the long-term preservation of dog semen, especially if the transport can be organized in 24 h.

Spermatozoa Survival in Egg Yolk-Based and Soybean-Based Extenders at Ambient and Chilling Temperature in Domestic Turkeys (Meleagris gallopavo).

Populations of many galliform species have declined mainly due to habitat loss and over-hunting, notably the Congo peacock, which has been classified as a vulnerable species by the International Union for Conservation of Nature (IUCN). The domestic turkey, being a species of least concern, which has been reported to be closely related to peacocks, could serve as a model for the optimization of assisted reproductive technologies for the Congo peacock. This study was aimed at developing a suitable turkey semen extender for artificial insemination in field conditions. Semen was collected using the dorso-abdominal massage technique from seven turkey toms and analyzed. Ejaculates with >70% motility and >80% live spermatozoa were pooled and divided into four aliquots (four treatments). Each of the four treatments was extended in a soybean-based extender or an egg yolk-based extender, with or without L-ascorbic acid. Two liquid preservation protocols (ambient temperature (35 °C) and chilled (4 °C)) were employed, and quality parameters including motility, viability and morphology were evaluated. The results show that the two extenders were similar with regard to semen quality parameters, and L-ascorbic acid supplementation of the turkey semen extenders improved semen quality during liquid storage.

Canine Spermatozoa-Predictability of Cryotolerance.

Markers of freezability allow the selection of ejaculates of good freezability. So far, most investigations were conducted in boars, bulls, rams and horses, with high economic interests triggering the efforts. The progress in dogs is comparably slow. A critical evaluation of the methods requires consideration of practicability, with most labs not even possessing a computer assisted sperm analyser (CASA); furthermore, small canine ejaculates mostly do not allow the use of large semen volumes. In dogs, modern markers of freezability no longer assess single membrane constituents or seminal plasma components but comprise tests of cell functionality and adaptability, energy metabolism, cluster analyses of kinetic and morphometric parameters, as well as DNA intactness. Identification of the most efficient combination of tests seems useful. At present, examination by CASA combined with cluster analysis of kinetic subgroups, JC-1 staining and COMET assay or staining with toluidine blue seem most appropriate; however, cell volumetry and other functional tests deserve better attention. A better understanding of spermatozoa energy metabolism might reveal new markers. This review focuses on the requirements and markers of freezability of canine semen, highlighting potential future candidates.

Fighting Like Cats and Dogs: Challenges in Domestic Carnivore Oocyte Development and Promises of Innovative Culture Systems.

In vitro embryo production in cats and dogs still presents some challenges, and it needs to be optimized to transfer efficient protocols to related wild, endangered species. While the chemical composition of culture media has been the focus of several studies, the importance of culture substrates for oocyte and embryo culture has often been neglected. Traditional in vitro systems, i.e., two-dimensional cultures, do not resemble the physiological environments where cells develop, and they may cause morphological and functional alterations to oocytes and embryos. More modern three-dimensional and microfluidic culture system better mimic the structure and the stimuli found in in vivo conditions, and they could better support the development of oocytes and embryos in vitro, as well as the maintenance of more physiological behaviors. This review describes the different culture systems tested for domestic carnivore reproductive cells along the years, and it summarizes their effects on cultured cells with the purpose of analyzing innovative options to improve in vitro embryo production outcomes.

Maturation and fertilization of African lion (Panthera leo) oocytes after vitrification.

The African lion is an excellent model species for the highly endangered Asiatic lion. African lions reproduce well in zoos, leading to the fact that occasionally ovaries and testis are available for in-vitro experiments. We previously performed in-vitro maturation (IVM) and fertilization of lion oocytes and were able to produce advanced embryos after intracytoplasmic sperm injection (ICSI) with cryopreserved sperm. Here we examined whether our in-vitro method is also applicable after vitrification of immature oocytes. Oocytes of four lionesses (5-7 years old) were obtained after euthanasia and immediately processed on site. Half of the oocytes (n = 60) were subjected to IVM for a total of 32-34 h at 39 °C, 5% CO2 and humidified air atmosphere. The second group (59 oocytes) was vitrified instantly using the Cryotop method. Following 6 days of storage in liquid nitrogen, oocytes were warmed and subjected to IVM as well. Mature oocytes of both groups were fertilized with frozen-thawed African lion sperm using ICSI. Maturation rate was 55% and 49.2% for the control and vitrified group, respectively. In the control group, three oocytes cleaved and another three were arrested at the pronuclei stage. Due to the low fertilization result, a sperm sample of another male was used for the vitrified group. Of the vitrified oocytes 7 cleaved and 9 more oocytes stopped at pronuclei stage. All embryos of the vitrified group did not develop beyond 4 cell stage. This is the first time that African lion in-vitro-derived embryos have been produced following oocyte vitrification.

Ovary cold storage and shipment affect oocyte yield and cleavage rate of cat immature vitrified oocytes.

In feline species, cooled transport of ovaries can be employed without detrimental effects to retrieve immature oocytes intended for in vitro embryo production purposes. Indeed, this is the most common way to collect gametes from gonads of wild, valuable animals after they die or are castrated far from specialized laboratories. However, fresh retrieved gametes are generally used, and their cryosensitivity is not known. This study employed ovariectomy-derived domestic cat gonads as a model for wild felids, and aimed to compare the yield and developmental competence of Cryotop-vitrified oocytes (VOs) collected and cryopreserved right after ovary excision (In loco-VOs) or after 24 h cooled transport of ovaries (Shipped-VOs). The number of collected oocytes was higher in In loco-VOs than in Shipped-VOs (mean ± SD: 8 ± 3.36 vs 5.6 ± 3.1, p = 0.05). In vitro embryo production resulted in similar maturation (35% for both vitrified groups, p = 1) and fertilization rates (In loco-VOs: 29.1%; Shipped-VOs: 22.2%; p = 0.295), but showed a difference in cleavage (In loco-VOs: 25.6%; Shipped-VOs: 14.5%; p = 0.0495). No differences were found in further embryo development. Taken together, results suggested that delayed oocyte vitrification after cooled transport of organs was feasible and allowed embryo development. However, the number of collected oocytes and the cleavage rate of matured oocytes were higher when oocyte vitrification was performed without delay after ovary excision, and this should be considered in gamete conservation programs for endangered felids.

Inhibition of Apoptotic Pathways Improves DNA Integrity but Not Developmental Competence of Domestic Cat Immature Vitrified Oocytes.

Being a model for endangered wild felids, cryopreservation protocols for domestic cat oocytes are under continuous development. Immature vitrified oocytes (VOs) are a valuable resource for fertility preservation programs, but they often degenerate after warming and their in vitro development is poor. Since the exact mechanisms are not clear, this study assessed whether vitrification might trigger two apoptotic markers (DNA fragmentation and caspase activity, Experiment I) and the effects of a chemical inhibitor (i.e., the pan-caspase inhibitor Z-VAD-FMK) on the same markers (Experiment II) and on VOs in vitro development (Experiment III). The overarching aim was to check whether apoptosis inhibition might be a strategy to improve cat oocytes cryotolerance. In Experiment I, vitrification induced DNA fragmentation and increased caspase activity in VOs incubated for 24 h after warming (DNA fragmentation: 59.38%; caspase activity: 414.6 ± 326.8) compared to a fresh control (9.68%; 199.6 ± 178.3; p = 0.02). In Experiment II, the addition of Z-VAD-FMK to vitrification-warming and incubation media decreased DNA fragmentation and caspase activity (8.82%; 243.7 ± 106.9) compared to control (untreated) VOs (69.44%; 434.5 ± 248.3; p < 0.001). In Experiment III, Z-VAD-FMK brought maturation rates of treated VOs close to those of fresh oocytes (53.13 and 65.38%, respectively, p = 0.057), but there were no differences in VOs embryo development (cleavage rates; Z-VAD-FMK-treated VOs: 34.38%; control VOs: 31.78%; p = 0.69). In summary, vitrification increased apoptotic markers in cat VOs, and while Z-VAD-FMK was able to hinder DNA damage and caspase activity, its addition was not determinant for embryo development. To make the best use of VOs, other oocyte in vitro maturation and embryo culture strategies, such as the addition of other inhibitors or their prolonged use, should be investigated.

Minimum Volume Vitrification of Immature Feline Oocytes.

In wild animals' conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator's skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.