Vitreous rebleeding rate after vitrectomy or phacovitrectomy in patients with proliferative diabetic retinopathy: A retrospective study.

BACKGROUND: The most common complication after vitrectomy is the rebleeding in vitreous cavity. It is important to detect the different factors that can increase the vitreous rebleeding rate in these patients. OBJECTIVE: To carry out a retrospective review of the rate of vitreous rebleeding after vitrectomy or phacovitrectomy. METHOD: Retrospective, descriptive and comparative study of patients with a diagnosis of proliferative diabetic retinopathy with phacovitrectomy or vitrectomy procedure. Personal background data, type of surgical intervention and grade of the surgeon who carried out the procedure were obtained. RESULTS: 1227 files of diabetic patients with vitrectomy or phacovitrectomy were reviewed. 65% presented systemic arterial hypertension. The average glomerular filtration rate was 63.50 (±32.36) ml/min/1.73 m2 and glycosylated hemoglobin (HBA1C) of 8% (4.6 to 15%). In the comparison of variables, a significant difference in the rate of vitreous rebleeding was obtained comparing phacovitrectomy with vitrectomy. (p = 0.003), in the relationship between vitrectomy with vitreous rebleeding, an odds ratio of 1.44 was obtained. CONCLUSION: The results obtained show a lower rate of rebleeding in patients undergoing phacovitrectomy in patients with proliferative diabetic retinopathy.

5-Fluorouracil drug delivery system based on bacterial nanocellulose for colorectal cancer treatment: Mathematical and in vitro evaluation.

Colorectal cancer (CRC) is the third most common worldwide. Its treatment includes adjuvant chemotherapy with 5-fluorouracil (5FU) administered intravenously. 5FU is an antineoplastic drug of the fluoropyrimidines group, widely used in the treatment of solid tumors, mainly CRC. Nevertheless, it causes several adverse effects and poor effectiveness due to its short half-life. This work aimed to employ bacterial nanocellulose (BNC) as an encapsulation material for the oral administration of 5FU. First, the adsorption phenomena were analyzed by isotherms, thermodynamic parameters, and kinetic models. Then, encapsulation was carried out using spray-drying, and encapsulated 5FU desorption profiles were assessed in simulated fluids. The biological behavior was evaluated on colon cancer SW480 and SW620 cell lines. As result, it was found that at 25 °C a monolayer of 5FU was formed and the process showed to be the most spontaneous one. In the characterization of the nanocapsules, important changes were detected by the presence of 5FU. The delivery in the colon corresponded to a controlled release behavior. The in vitro assay indicated an improvement in the toxicity effect of the drug and its mechanism of action. Accordingly, BNC is a promising biomaterial for the development of a colon drug delivery platform of 5FU.

Global diagnosis of nitrate pollution in groundwater and review of removal technologies.

Clean water and sanitation for the world population is one of the most important challenges established by the Sustainable Development Goals of the United Nations since worldwide, one in three people do not have access to safe drinking water. Groundwater, one of the main sources of fresh water, has been considerably damaged by human activities. Nevertheless, while numerous plants are globally aimed at removing pollutants from surface waters, a much scarcer number of facilities have focused on groundwater remediation. Nowadays, there is increasing concern about the presence of nitrates (NO3-) in groundwaters as a consequence of the intensive use of fertilizers and other anthropogenic sources, such as sewage or industrial wastewater discharge. In this context, the selection and development of highly effective and low-cost solutions for the sustainable management of groundwater resources need to be addressed. Thus, this work collects data from the literature regarding the presence of nitrates in groundwater, and, simultaneously, it reviews the main alternatives available to remove NO3- from groundwater sources. A total of 292 sites have been analyzed categorized by continents, carefully discussing the possible origins of nitrate pollution. In addition, a discussion is carried out of the different technologies currently employed to treat groundwater, highlighting the progress made and the main challenges to be overcome. Finally, the review gathers the data available in the literature for nitrate treatment plants at full-scale.

Variation in resistance to oxidative stress in Oregon-(R)R-flare and Canton-S strains of Drosophila melanogaster.

All aerobic organisms are susceptible to damage by reactive oxygen species (ROS). ROS-induced damage has been associated with aging and diseases such as metabolic syndrome and cancer. However, not all organisms develop these diseases, nor do they age at the same rate; this is partially due to resistance to oxidative stress, a quantitative trait attributable to the interaction of factors including genetics and environmental. Drosophila melanogaster represents an ideal system to study how genetic variation can affect resistance to oxidative stress. In this work, oxidative stress (total and mitochondrial ROS), antioxidant response, and Cap 'n' collar isoform C and Spineless gene expression, one pesticide resistant (Oregon R(R)-flare) and wild-type (Canton-S) strains of D. melanogaster, were analyzed to test resistance to basal oxidative stress. ROS, catalase, and superoxide dismutase were determined by flow cytometry, and Cap 'n' collar isoform C and Spineless expression by qRT-PCR. The intensity of oxidative stress due to the pro-oxidant zearalenone in both was evaluated by flow cytometry. Data confirm expected differences in oxidative stress between strains that differ in Cyp450s levels. The Oregon (R)R-flare showed greater ROS, total and mitochondrial, compared to Canton-S. Regarding oxidative stress genes expression Cap 'n' collar isoform C and Spineless (Ss), Oregon R(R)-flare strain showed higher expression. In terms of response to zearalenone mycotoxin, Canton-S showed higher ROS concentration. Our data show variation in the resistance to oxidative stress among these strains of D. melanogaster.

Synchronous bilateral breast cancer treated with a 3-week hypofractionated radiotherapy schedule: clinical and dosimetric outcomes

Background and purpose Synchronous bilateral breast cancer (SBBC) accounts for 1–3.5% of breast cancer patients. The aim of this study was to evaluate dosimetric issues, clinical outcomes, and acute toxicities for SBBC patients receiving synchronous bilateral hypofractionated radiotherapy (SBHRT) and to compare them with patients treated with synchronous bilateral normofractionated RT schedule (SBNRT). Materials and methods From April 2016 to March 2020, 39 SBBC patients were referred to our institution. Patients were divided according to their prescription dose: Group A: 50 Gy/25fx (fractions), B: 60–64 Gy/25fx, C: 40.05 Gy/15fx; D: 48 Gy/15fx. Toxicity was evaluated using Common Terminology Criteria for Adverse Events (CTCAE)v.5.0. Results 34 patients were finally evaluated. Median follow-up was 24 months for NF schedule and 9 months for HF schedule. In the HF schedule, no acute side-effects > G2 were observed and no dermatitis was reported in 6th month´s assessments. 95% of patients have no evidence of disease and only 1 patient presented local relapse in the first mammography after RT. No distant failures or deaths were observed. Regarding dosimetric issues, the inter-patient average Dmean for the heart was: Group A: 5.0 Gy (4.6–5.5), Group B: 4.4 Gy (4.1–5.4), Group C: 4.8 Gy (4.5–5.1) and Group D: 5.3 Gy (4.4–5.6). For the lungs, the inter-patient average Dmean was: Group A: 10.8 Gy (9.8–12.2), Group B: 11.5 Gy (11.3–12), Group C: 9.8 Gy (9.3–10.5) and Group D: 10.5 Gy (10–11.3). Conclusions This is the first study reporting the safety, feasibility, and tolerability of 40.05 Gy/15fx over 3 weeks for the treatment of SBBC patients. Further study with larger accrual is mandatory (AU)

Primary and secondary retinal capillary haemangioma in Mexico.

A series is presented of sixteen cases of retinal capillary haemangioma (RCH) from consecutive patients at an ophthalmology teaching hospital in Mexico City. There were seven primary haemangioblastomas, and nine due to von Hippel-Lindau disease (VHL). All cases associated with VHL already had systemic manifestations, such as, cerebellar, medullary and renal tumours. Treatment of capillary haemangiomas must be individualised, based on several factors, including the number of lesions, exudation, or presence of retinal detachment. A multidisciplinary approach is essential.

Statistical control of processes applied to geometric uncertainties for CTV expansion margins determination in prostate cancer patients treated with VMAT: a prospective study in 57 patients

Purpose To study the control graphs applicability for the geometric uncertainties of VMAT treatments in prostate cancer patients, and their use to verify the hypothesis of the data obtained randomness, to apply the margins of Van Herk expression. Material and methods During the first 5 days of treatment, and then once a week, a Kv CBCT was performed, compared with the simulation CT and adjusted the displacements, to determine the inter-fraction errors. Immediately after radiation therapy, another CBCT was performed (for intra-fraction errors). With these data, the X, R position control charts have been made. The patients, not maintained the deviations within the charts control limits, were called “anomalies”. Then, we compared the deviations and margins calculated with the van Herk expression for all patients and for those without anomalies. Results The margins determined show appreciable differences if there were calculated for the total set of patients or for the set of them without anomalies in the control charts. For the overall set of patients, the lateral, longitudinal, and vertical margins were 0.45 cm, 0.52 cm, 0.56 cm, while for the set of patients without anomalies were 0.29 cm, 0.35 cm, and 0.38 cm. Conclusions The use of control charts allows tracking geometric deviations both inter and intra-fraction, variability real-time control and to detect situations in which it can change for non-random reasons, and require immediate investigation. Maintaining geometric deviations in the control state decreases the margins needed to administer a high dose to CTV in a high percentage of cancer prostate patients (AU)

Hemangioma capilar de retina primario y secundario en México / Primary and secondary retinal capillary haemangioma in Mexico

Se presenta una serie de 16 casos de hemangioma capilar retiniano de pacientes valorados en un hospital oftalmológico de enseñanza en la Ciudad de México: 7 de ellos eran hemangioblastomas primarios y 9 eran secundarios a enfermedad de von Hippel-Lindau. Todos los casos relacionados con esta enfermedad presentaban en ese momento manifestaciones sistémicas como: tumores cerebelosos, medulares o renales. El tratamiento para el hemangioma capilar retiniano debe ser individualizado con base en diversos factores como el número de lesiones, la exudación o la presencia de desprendimiento de retina asociado. Se debe de contar con un abordaje multidisciplinario (AU)

A series is presented of 16 cases of retinal capillary haemangioma from consecutive patients at an ophthalmology teaching hospital in Mexico City. There were 7 primary haemangioblastomas, and 9 due to von Hippel-Lindau disease. All cases associated with this disease already had systemic manifestations, such as cerebellar, medullary and renal tumours. Treatment of capillary haemangiomas must be individualised, based on several factors, including the number of lesions, exudation, or presence of retinal detachment. A multidisciplinary approach is essential (AU)

Trombosis venosa ovárica posparto: una causa inusual de fiebre puerperal / Postpartum ovarian venous thrombosis: An unusual cause of puerperal fever

La trombosis de la vena ovárica posparto es una complicación rara e impredecible y que requiere un alto índice de sospecha clínica, cuyo diagnóstico y manejo precoces son esenciales, requiriendo un abordaje multidisciplinar, para evitar cirugías innecesarias y posibles complicaciones graves. Su presentación clínica habitual suele ser fiebre persistente y dolor abdominal, generalmente en la primera semana posparto. Revisamos 3 casos clínicos representativos, diagnosticados en nuestro centro entre 2014 y 2019, abordando los factores predisponentes, diagnóstico, manejo y evolución de estas pacientes.(AU)

Postpartum ovarian vein thrombosis is a rare and unpredictable complication that requires a high index of clinical suspicion. Its early diagnosis and management, with a multidisciplinary approach, are essential in order to avoid unnecessary surgeries and possible serious complications. Its usual clinical presentation is often persistent fever and abdominal pain, generally in the first week postpartum. A report is presented on three representative cases that were diagnosed in our centre between 2014 and 2019, presenting predisposing factors, diagnosis, management, and outcome of these patients.(AU)

Synchronous bilateral breast cancer treated with a 3-week hypofractionated radiotherapy schedule: clinical and dosimetric outcomes.

BACKGROUND AND PURPOSE: Synchronous bilateral breast cancer (SBBC) accounts for 1-3.5% of breast cancer patients. The aim of this study was to evaluate dosimetric issues, clinical outcomes, and acute toxicities for SBBC patients receiving synchronous bilateral hypofractionated radiotherapy (SBHRT) and to compare them with patients treated with synchronous bilateral normofractionated RT schedule (SBNRT). MATERIALS AND METHODS: From April 2016 to March 2020, 39 SBBC patients were referred to our institution. Patients were divided according to their prescription dose: Group A: 50 Gy/25fx (fractions), B: 60-64 Gy/25fx, C: 40.05 Gy/15fx; D: 48 Gy/15fx. Toxicity was evaluated using Common Terminology Criteria for Adverse Events (CTCAE)v.5.0. RESULTS: 34 patients were finally evaluated. Median follow-up was 24 months for NF schedule and 9 months for HF schedule. In the HF schedule, no acute side-effects > G2 were observed and no dermatitis was reported in 6th month´s assessments. 95% of patients have no evidence of disease and only 1 patient presented local relapse in the first mammography after RT. No distant failures or deaths were observed. Regarding dosimetric issues, the inter-patient average Dmean for the heart was: Group A: 5.0 Gy (4.6-5.5), Group B: 4.4 Gy (4.1-5.4), Group C: 4.8 Gy (4.5-5.1) and Group D: 5.3 Gy (4.4-5.6). For the lungs, the inter-patient average Dmean was: Group A: 10.8 Gy (9.8-12.2), Group B: 11.5 Gy (11.3-12), Group C: 9.8 Gy (9.3-10.5) and Group D: 10.5 Gy (10-11.3). CONCLUSIONS: This is the first study reporting the safety, feasibility, and tolerability of 40.05 Gy/15fx over 3 weeks for the treatment of SBBC patients. Further study with larger accrual is mandatory.

Statistical control of processes applied to geometric uncertainties for CTV expansion margins determination in prostate cancer patients treated with VMAT: a prospective study in 57 patients.

PURPOSE: To study the control graphs applicability for the geometric uncertainties of VMAT treatments in prostate cancer patients, and their use to verify the hypothesis of the data obtained randomness, to apply the margins of Van Herk expression. MATERIAL AND METHODS: During the first 5 days of treatment, and then once a week, a Kv CBCT was performed, compared with the simulation CT and adjusted the displacements, to determine the inter-fraction errors. Immediately after radiation therapy, another CBCT was performed (for intra-fraction errors). With these data, the X, R position control charts have been made. The patients, not maintained the deviations within the charts control limits, were called "anomalies". Then, we compared the deviations and margins calculated with the van Herk expression for all patients and for those without anomalies. RESULTS: The margins determined show appreciable differences if there were calculated for the total set of patients or for the set of them without anomalies in the control charts. For the overall set of patients, the lateral, longitudinal, and vertical margins were 0.45 cm, 0.52 cm, 0.56 cm, while for the set of patients without anomalies were 0.29 cm, 0.35 cm, and 0.38 cm. CONCLUSIONS: The use of control charts allows tracking geometric deviations both inter and intra-fraction, variability real-time control and to detect situations in which it can change for non-random reasons, and require immediate investigation. Maintaining geometric deviations in the control state decreases the margins needed to administer a high dose to CTV in a high percentage of cancer prostate patients.

Factors Affecting Embryo Recovery Rate, Quality, and Diameter in Andalusian Donkey Jennies.

Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (PFR; p = 0.040) and embryo recovery (ERR; p < 0.05) as well as embryo quality (p = 0.004). ERR was also affected by the number of flushings (p < 0.001), donor age (p < 0.05), successive cycle within donor (p < 0.001), and jacks (p < 0.05). Number of flushings (p < 0.001) and jack (p < 0.05) had a significant effect on PFR, whereas the day of flushing influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos (p < 0.001). The number of flushings significantly influenced the diameter (p = 0.038) and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different between herds (p = 0.020). The factors influencing the success of this assisted reproductive technique were donor jenny, donor age, successive cycle within donor, day of flushing, number of flushings, and jack. The identification of these key points is crucial to achieve a higher efficiency of embryo transfer and vitrification processes, before considering their application in the conservation of endangered donkey breeds.

One-step warming does not affect the in vitro viability and cryosurvival of cryotop-vitrified donkey embryos.

The objective of this study was to compare the effects of two warming protocols (three-step vs. one-step dilution) on embryo quality, post-warming embryo survival and embryo cell viability of donkey embryos vitrified by the Cryotop method. Twenty, Day 7-8, grade 1-2 donkey embryos were measured, morphologically evaluated and vitrified using the Cryotop technique. Embryos were then randomly warmed using two different warming procedures: (i) W3 (three-step dilution; n = 11): embryos were warmed in 1 M, 0.5 M and 0 M sucrose, and (ii) W1/0.5 (one-step dilution; n = 9): embryos were warmed directly in 0.5 M sucrose. After 3 and 24 h of warming, the embryos were measured and evaluated for their morphology, developmental stage and viability (Propidium Iodide-Hoechst 33,342 dyes). Although both treatments decreased embryo quality after warming (P < 0.05), no significant differences (P > 0.05) were observed between protocols in terms of post-warming embryo quality, diameter and embryo survival. Greater percentages of dead cells (P < 0.001) were observed when embryos were warmed directly in 0.5 M sucrose (one-step dilution) when compared to the three-step protocol. The percentage of ruptured embryos was 27.3% and 0% in W3 and W1/0.5 protocols (P = 0.0893), respectively. In conclusion, warming Cryotop-vitrified donkey embryos directly in 0.5 M sucrose had no negative effects on embryo quality and post-warming embryo survival. Moreover, one-step protocol may help to prevent rupture when donkey embryos warmed directly in 0.5 M sucrose. These results observed in vitro must be verified by embryo transfer.

Nano-depletion of acrosome-damaged donkey sperm by using lectin peanut agglutinin (PNA)-magnetic nanoparticles.

Lectin is considered as a suitable biomarker for nano-depletion of acrosome-damaged sperm. The aim of this study was to synthetize magnetic nanoparticles (MNPs) coated by peanut (Arachis hypogaea) agglutinin lectin (PNA) and investigate its beneficial effect in improving of sperm characteristics. MNPs were obtained by co-precipitation method, functionalized with chitosan and coated by PNA at a concentration of 0.04 mg/mL. Semen was frozen either with glycerol-based or sucrose-based extenders. Frozen-thawed straws from five donkeys (three ejaculates per donkey) were incubated with lectin-MNPs (2 mg/mL), and then exposed to an external magnet enabling the non-bound sperm to be collected as nanopurified sperm. Sperm were evaluated post-thawing (control) and after nanopurification for motility, plasma membrane integrity, acrosome integrity, morphology, DNA fragmentation and concentration. The statistical analyses were extended to investigate the correlation between the initial quality of the frozen-thawed semen samples and the effect of nanopurification after thawing. The obtained MNPs were biocompatible to the sperm and significantly improved the progressive motility (P < 0.05) for the glycerol nanopurified group (43.08 ± 3.52%) in comparison to control (33.70 ± 2.64%). Acrosome-damaged sperm were reduced (P < 0.05) in both nanopurified groups (19.92 ± 2.69 for G and 21.57 ± 2.77 for S) in comparison to control (36.07 ± 3.82 for G and 35.35 ± 3.88 for S). There were no significant changes in sperm morphology and membrane integrity after nanopurification. The average sperm recovery after nanopurification was 80.1%. Sperm quality index was significantly higher (P < 0.001) in nanopurified groups regardless of the initial quality of the frozen thawed semen samples. However, in the high sperm quality group, nanopurification significantly improved the progressive motility and membrane integrity besides the increasing of acrosome-intact sperm. Sperm nanopurification using lectin-magnetic nanoparticles can be considered as a suitable method to reduce the proportion of acrosome-damaged sperm and to increase the quality of frozen thawed donkey semen.

Not every pigmented tumour is melanoma: Adenoma of the ciliary body pigment epithelium. / No todo tumor hiperpigmentado es melanoma: adenoma del epitelio pigmentario del cuerpo ciliar.

Differential diagnosis between a melanoma and a pseudomelanoma requires many years of experience and ocular studies, and requires paying attention to the smallest details. The case is presented of a 57 year-old male with an adenoma of the ciliary body pigment epithelium that was treated with an enucleation of the eye due to a suspicion of a choroidal melanoma.

Seasonal variations in sperm DNA fragmentation and pregnancy rates obtained after artificial insemination with cooled-stored stallion sperm throughout the breeding season (spring and summer).

The aim of this study was to assess seasonal variations during different periods of the breeding season (spring and summer) on stallion sperm DNA fragmentation and in vivo fertility associated with cooled-stored semen samples. Ejaculates were collected from eleven stallions and assessed for sperm motility (assessed by computer-assisted sperm analysis) and plasma membrane integrity (evaluated under fluorescence microscopy). Sperm DNA fragmentation (evaluated by the Sperm Chromatin Dispersion test) was assessed in cooled-stored semen at 5 °C for up to 24 h. Artificial insemination was performed throughout the breeding season. Mares were inseminated with cooled-stored semen (up to 24 h) every other day until ovulation. Pregnancy rates per cycle were determined detecting the embryonic vesicle by ultrasonography fifteen days after ovulation. Values (mean ± SD) for progressive sperm motility were significantly higher (P < 0.05) in spring (53.57 ± 9.97%) in comparison to summer (41.37 ± 10.81%). No significant differences in plasma membrane integrity were found between seasons (P > 0.05). Sperm DNA fragmentation was significantly lower (P < 0.01) in spring in comparison to summer after 0h (4.81 ± 1.87% vs. 8.77 ± 5.78%), 6h (9.00 ± 3.19% vs. 18.73 ± 8.22%) and 24h (14.6 ± 4.13% vs. 30.14 ± 9.85%) of cooled-storage. Pregnancy rates per cycle were also significantly higher (P < 0.01) in spring (50%) in comparison to summer (37%). There was a moderate negative relationship between positive pregnancies and sperm with fragmented DNA (r = - 0.619; P < 0.001). Semen samples associated with moderate fertility levels (Pregnancy rate < 50%) showed a higher percentage of sperm with fragmented DNA compared to samples obtaining higher fertility levels. In conclusion, seasonal variations were found during the breeding season, obtaining lower sperm DNA fragmentation and higher pregnancy rates in spring. Additionally, samples with the highest proportion of sperm with fragmented DNA showed the lowest fertility levels throughout the breeding season.

Potential formation of PCDD/Fs in triclosan wastewater treatment: An overall toxicity assessment under a life cycle approach.

Wastewater may contain a diverse group of unregulated pollutants known as emerging pollutants, such as pharmaceuticals and personal care products (PPCPs). Triclosan (TCS) is a personal care product widely used as an antiseptic or preservative in cosmetics, hand wash, toothpaste and deodorant soaps. Advanced oxidation processes (AOPs) have been used as effective and alternative treatments for complex wastewater. However, an important criterion for the assessment of AOPs and their operation conditions could be the potential formation of new toxic secondary products, such as polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), especially when emerging pollutants are present in the media. If these are omitted from environmental management studies, the real environmental impacts of a WWTPs (wastewater treatment plants) may be underestimated. Consequently, the current study aims to evaluate the environmental impacts derived from electrooxidation (EOX), one of the most effective oxidation technologies, of emerging pollutants using Life Cycle Assessment. The analyses were performed for the treatment of effluents containing TCS, firstly without considering the formation of PCDD/Fs and, thereafter, considering the effects of these compounds. Total toxicity, calculated through different methods and corresponding impact factors, were evaluated for each stage of the process when different electrolytes are used, including PCDD/Fs formation. Finally, a sensitivity analysis was carried out to study i) the effect of the TCS initial concentration on the environmental impacts associated to ecotoxicity for the different life cycle methods and ii) the influence of changing the organic pollutant on PCDD/Fs formation employing 2-chlorophenol (2-CP). As a result, LCIA methods demonstrate that they are not fully adapted to the computation of PCDD/Fs in the water compartment, since only 2,3,7,8-tetraclorodibenzo-p-dioxina (2,3,7,8-TCDD) is present as a substance in the impact categories assessed, ignoring the remaining list of PCDD/Fs.

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.

Application of embryo biopsy and sex determination via polymerase chain reaction in a commercial equine embryo transfer program in Argentina.

Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active embryo transfer program. We evaluated results on biopsy of 459 embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped embryos biopsied ≥20h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a positive genomic (g) DNA result was significantly higher for biopsies from large blastocysts (96%) than from smaller embryos (70-85%). In total, 38% of biopsies were positive for Y chromosome DNA (Y-DNA) and were diagnosed as male. Subsequently, 95% of Y-DNA-positive embryos were confirmed as male and 78% of Y-DNA-negative embryos were confirmed as female. The accuracy of prediction of female (Y-DNA negative) was significantly higher when the biopsy sample was probed for Y-DNA only compared with probing for both gDNA and Y-DNA. We estimate that by transferring only Y-DNA-negative embryos, 3% of potential female pregnancies may have been lost, and production of male pregnancies was reduced by 72%.

Cryopreservation of Andalusian donkey (Equus asinus) spermatozoa: Use of alternative energy sources in the freezing extender affects post-thaw sperm motility patterns but not DNA stability.

The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the freezing extender improved total and progressive sperm motility (P < 0.05) and amplitude of lateral head displacement (P < 0.01), but it reduced the values for other sperm motility variables compared with glucose (P < 0.001). The use of fructose resulted in a reduction in values for most CASA variables (P < 0.05), whereas addition of Equex paste did not have any beneficial effect on values for these variables (P > 0.05). Glucose was more effective in maintaining sperm morphology (P < 0.05), while there was no beneficial effect with the addition of Equex paste (P > 0.05). Supplementation of fructose and Equex paste in the freezing extender decreased plasma membrane integrity (P < 0.05) as compared with glucose, but there were no differences between treatments for acrosome and DNA integrity (P > 0.05), even after 24 h of incubation. The use of different sugar sources in the extender could affect the in vitro post-thaw quality of cryopreserved donkey spermatozoa, with sorbitol being an interesting alternative for improving the sperm quality. Results of the present study indicate the use of Equex paste could negatively affect post-thaw outcomes for sperm viability in this species.