Current status of fertility preservation in a Spanish tertiary public hospital: multidisciplinary approach and experience in over 1500 patients.

PURPOSE: Currently, 15% of gynaecological and 9% of haematological malignancies are diagnosed before the age of 40. The increased survival rates of cancer patients who are candidates for gonadotoxic treatments, the delay in childbearing to older ages, and the optimization of in vitro fertilisation techniques have all contributed to an increased interest in fertility preservation (FP) treatments. This study reviews the experience of the Fertility Preservation Programme (FPP) of a tertiary public hospital with a multidisciplinary approach. METHODS: This retrospective study included all the available (FP) treatments, performed in patients of childbearing age between 2006 and 2022. RESULTS: 1556 patients were referred to the FPP: 332 oocyte vitrification cycles, 115 ovarian cortex cryopreservation with 11 orthotopic autotransplantations, 175 gonadotropin-releasing hormone (GnRH) agonist treatments, 109 fertility-sparing treatments for gynaecological cancer, and 576 sperm cryopreservation were performed. Malignancy was the main indication for FP (the main indications being breast cancer in women and haematological malignancies in men), although non-oncological pathologies, such as endometriosis and autoimmune diseases, have increased in recent years. Currently, the most widely used FP technique is oocyte vitrification, the increase of which has been associated with a decrease in the use of cortex CP and GnRH agonists. CONCLUSIONS: The increase in FP treatment reflects the implementation of reproductive counselling in oncology programmes. A multidisciplinary approach in a tertiary public hospital allows individualised FP treatment for each patient. In recent years, there has been a change in trend with the introduction of new indications for FP and a change in techniques due to their optimisation.

Critérios e metodologias para produzir sêmen de caprinos e ovinos em nível de fazenda / The criteria and methodologies used to produce ram and goat semen on the farm

A criopreservação de sêmen é uma importante estratégia para o armazenamento de material genético de reprodutores ovinos e caprinos, considerados superiores do ponto de vista fenotípico, mas especialmente, pelos seus méritos genéticos. Hoje no Brasil apenas uma central regulamentada pelo MAPA está ema atividade, o que significa que existe pouca dose de sêmen comercial à disposição para programas de melhoramento genético. Programas de banco de sêmen podem ser realizado nas fazendas, entretanto, tem seu uso limitado a propriedade onde está o reprodutor. Neste caso, apesar de maiores limitações de estrutura e equipamentos para a execução do serviço, o domínio das variações da técnica de congelação de sêmen e o cuidado com as questões sanitárias dos reprodutores possibilitará ao Médico-Veterinário a execução do serviço com níveis adequados de segurança e qualidade das doses de sêmen produzidas. O objetivo deste estudo é apresentar e discutir critérios e metodologias para congelação de sêmen de pequenos ruminantes em diferentes condições, fruto de diversas experiências e trabalhos desenvolvidos por nosso grupo e outros, nos últimos vinte anos, e que tem possibilitado resultados satisfatórios.(AU

Sperm cryopreservation is an important strategy for the storage of genetic material from ovine and goat breeders, considered superior from the phenotypic point of view, but especially for their genetic merits. Today in Brazil only one center regulated by the Ministry of Agriculture, Livestock and Supply is in operation, which means that there is little dose of commercial semen available for genetic improvement programs. Semen bank programs can be carried out on farms; however, their use is limited to the property where the breeder is located. In this case, despite greater limitations of structure and equipment for the execution of the service, mastering variations in the semen freezing technique and taking care of the health issues of the breeders will enable the Veterinarian to perform the service with adequate levels of safety and quality of semen doses produced. This study aims to present and discuss criteria and methodologies for freezing semen from small ruminants under different conditions, the result of several experiences and works carried out by our group and others, in the last twenty years, which have enabled satisfactory results.(AU))

Relationship between the seasonal changes in plasma testosterone and thyroxine concentrations with sperm cryoresistance in Gabon bucks.

Gabon buck is a breed with little marked seasonality in our latitude (Uruguay, 35° SL). The role of thyroid hormones on the regulation of their seasonal reproductive activity and sperm cryoresistance is unknown. Seasonal changes in testosterone concentration can affect sperm variables, but the influence of testosterone changes on sperm cryoresistance in other species determines that the recommended time for freezing sperm does not coincide with the period with greater sperm fresh quality. The objectives of the present work were to (i) describe the thyroxine seasonal pattern in bucks in a subtropical area, and its association with annual changes in sperm variables; (ii) relate the seasonal changes of testosterone and thyroxine concentrations with the sperm cryoresistance. For one year, semen of 10 adult Gabon bucks was collected by electroejaculation every two weeks. After sperm selection, the sample was frozen. Testosterone and thyroxine concentrations varied according to the month (P < 0.0001). Testosterone reached the greatest values in April (P < 0.0001) and May (P < 0.0001) and thyroxine reached minimum values (P < 0.0001) in the same months. During these months, a negative correlation ratio (CR) was found between testosterone concentration and CR-functional membrane (R = - 0.50; P < 0.0001). CR values for most sperm variables decreased during March-May, coinciding with the presence of maximum testosterone concentrations. In conclusion, high testosterone levels are associated with the worst sperm response to freezing-thawing process. Thyroxine concentrations have a strong seasonal pattern, but there was no relationship to sperm cryoresistance.

Phosphatidylcholine and L-acetyl-carnitine-based freezing medium can replace egg yolk and preserves human sperm function.

BACKGROUND: Conventional cryopreservation methods induce chemical and mechanical damage to the sperm membranes. The cryoprotectant potential of phospholipids of vegetal origin as soybean lecithin has been investigated as a substitute for egg yolk in diluents used for the cryopreservation of human spermatozoa. Therefore, the objective of this study was comparing the efficacy of a synthetic cryoprotectant supplemented with L-α-phosphatidylcholine (PC) and L-acetyl-carnitine (ANTIOX-PC) and the standard egg-based TEST-yolk buffer (TYB) in preserving sperm motility and chromatin quality in cryopreserved semen samples. METHODS: Prospective experimental study in which semen samples from 63 men with normal sperm motility and 58 men with low sperm motility were included and analyzed both before and after cryopreservation using ANTIOX-PC or TYB freezing media. Sperm quality was evaluated by routine semen analysis and DNA fragmentation index using the Terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: Differences in the post-thaw progressive motility and DNA fragmentation index were not detected between TYB and ANTIOX-PC cryoprotectants in both normal and low sperm motility groups (P>0.05). However, ANTIOX-PC medium retained higher non-progressive motility and lower percentage of immotile sperm when compared to TYB medium, resulting in a greater total motile sperm count (P<0.05), regardless baseline values of motility characteristic of the normospermic or asthenozoospermic samples. CONCLUSIONS: ANTIOX-PC medium was effective to protect human sperm during a freeze-thaw cycle compared to the TYB medium. A clinically relevant advantage in better preserving kinetic parameters as higher total motility and lower immotile post-thawed sperm from ANTIOX-PC, in normal and low motility semen samples, demonstrated the positive impact of phospholipid and antioxidant treatment on sperm cryotolerance with high potential for egg yolk lipids replacement and biosafety.

Effects of resveratrol in bull semen extender on post-thaw sperm quality and capacity for fertilization and embryo development.

Resveratrol, a potent antioxidant, can be an alternative semen extender constituent to protect spermatozoa against reactive oxygen species (ROS); however, effects on sperm quality post-thawing and sperm function is not well understood. This study, therefore, was conducted to investigate effects of resveratrol supplementation to semen extender on sperm quality post-thawing. Bull semen was cryopreserved using extenders not supplemented or supplemented with 0.05, 0.1, or 1 mM resveratrol. Supplementation of extender with resveratrol at 0.05 mM resulted in greater (P < 0.05) sperm progressive motility, average path velocity, straight linear velocity, linearity and straightness when compared with no or 1 mM supplementations. Furthermore, effects of 0.05 mM resveratrol supplementations on plasma membrane and acrosome integrity and sperm fertilization capacity using in vitro procedures were investigated. Supplementation of semen extender with resveratrol resulted in a greater (P < 0.05) proportion of frozen-thawed spermatozoa with an intact acrosome and plasma membrane. Results from in vitro fertilization studies indicated there were no differences (P> 0.05) when there was no supplementation or supplementation with 0.05 mM resveratrol on embryo development to the cleavage and blastocyst stages. In conclusion, addition of resveratrol to bull semen extender resulted in greater sperm quality post-thawing in a dose-dependent manner, with values for variables related to sperm quality being greater when there was resveratrol supplementation at the 0.05 mM concentration. Proportion of embryo developing to the cleavage and blastocyst stages after in vitro fertilization was not affected by resveratrol supplementation to semen extenders.

Evaluation of Chilled Dog Semen Extended With Sperm Activator.

Within modern biotechnology, different tools and methodologies have been developed to maximize canine semen conservation protocol to optimize reproductive results. In the last decades, the survival of chilled semen has been prolonged from 2 to 3 days with the first basic diluents, to 10-14 days with the modern extenders. However, their main limitation is that sperm quality decreases during cold storage. Sperm activators (SA) have been produced to provide the molecules necessary to maximize the sperm survival and quality with the aim to enhance fertility and prolificacy. In this study, the effect of commercial extender SA (Theriosolution® Canine AI extender -Chile-) was recorded by daily evaluation of chilled semen for 14 days. In this experiment, sperm-rich ejaculate fraction was collected from six adult healthy Neapolitan Mastiff dogs. The semen evaluation started immediately after collection (d0), and after that a next generation extender was added (d0) for every 24 h from d1 (with and without SA) to d14, to determine spermatozoa progressive motility, velocity of forward progression (VFP), morphology, and integrity of the spermatic membrane. The initial sperm concentration of extended semen was 417.3 ± 170.4 x 106/mL (mean ± SEM) with 85.89 ± 4.76% of MNS (morphologically normal sperm), 84.47 ± 5.22 % live sperm, and pH of 6.2 ± 2.8. The initial VFP was 3.83 ± 0.48, but after 1 min with SA, it rises to 4.45 ± 0.45 (P < 0.001). The sperm progressive motility parameter increases significantly (P < 0.05) in experimental trial, respect to control, starting to d2 at finish (except for d7). The VFP analysis significantly increases in experimental trial (P < 0.05) during most days of the study with the exclusion of d3 and d14. To evaluate the seminal characteristics over time, the experiment was divided into T1 (d0-d5), T2 (d6-d10), and T3 (d11-d14) (P < 0.001) in evaluation of morphology and membrane functionality. The MNS reached 70% at d10 and finally 65% at d14, being considered normal and possibly fertile. With Host-s, 65% of MNS were also achieved at d14. The presence of glucose and fructose in the diluents used for refrigeration can exert very important effects given the fact that metabolic routes have been found in both sugars, providing both different and complementing effects. It can be concluded that the use of SA prior to artificial insemination improves the quality of chilled semen significantly, although it does not reverse the effects of deterioration due to cellular metabolism over time.

Susceptibility of epididymal sperm against reactive oxygen species in dogs.

In order to add information to physiology of sperm maturation and help to underline future antioxidant supplementation treatment to epididymal sperm, the aim of this study was to evaluate susceptibility of caput, corpus and cauda epididymal sperm to different reactive oxygen species (ROS) in dogs. Epididymal sperm was separately collected from each segment (caput, corpus and cauda) and subjected to 4 different ROS-challenges: superoxide anion, hydrogen peroxide, hydroxyl radical, malondialdehyde (MDA) or maintained as control. After 30 min of incubation in each ROS, sperm was evaluated for sperm kinetics, plasma and acrossomal membrane integrity, mitochondrial activity and resistance to oxidative stress. Decreased total and progressive sperm motility and rapid velocity at epididymal corpus occurred after exposure to hydrogen peroxide, hydroxyl radical and MDA. However, for cauda epididymis, hydrogen peroxide and malondialdehyde promoted higher deleterious effect regarding sperm motility and velocity. Only at cauda epididymis MDA decreased sperm mitochondrial activity index and no kinetics alterations (motility or velocity) occurred after exposure to superoxide anion in corpus and cauda epididymis. In conclusion, corpus and cauda epididymal sperm are highly susceptible to deleterious effect of hydrogen peroxide, malondialdehyde and hydroxyl radical. In addition, epididymal canine sperm is relatively resistant to superoxide anion damage.

Antioxidants and their effect on the oxidative/nitrosative stress of frozen-thawed boar sperm.

In swine, the use of frozen-thawed boar sperm for artificial insemination remains a suboptimal reproductive technology. Among the negative effects of cryopreservation on sperm cells, it is worth highlighting that cryopreservation causes irreversible alterations in motility and components of the sperm membrane as a result of dramatic changes in temperature (cooling/freezing curve) and osmolality. In addition, freeze-thawing may induce oxidative stress and increase the generation of reactive oxygen species (ROS) and nitrogen reactive species (RNS). While boar sperm cryopreservation has been reported to increase lipid peroxidation and the intracellular levels of hydrogen peroxide, less research on its impact on RNS has been conducted. Furthermore, previous studies have investigated the effects of supplementing cryopreservation media with antioxidants to counteract the deleterious effects of ROS and RNS. Antioxidants of synthetic origin or natural extracts have been used, with some showing noticeable and positive effects on functional sperm parameters both in vitro and in vivo. The aim of this review is to provide an update on the effect of different molecules with antioxidant capacity on the function of cryopreserved boar sperm.

Protective effect of the superoxide dismutase mimetic MnTBAP during sperm vitrification process.

Sperm cryopreservation is widely used in assisted reproduction and male infertility therapy; however, it induces oxidative stress affecting sperm quality. This work evaluated the effect of the antioxidant MnTBAP during vitrification steps in human spermatozoa. First, the effect of MnTBAP on viability and ROS production was evaluated. Then, the spermatozoa were vitrified in straws with the vitrification, warming and post-warming incubation media separately supplemented with MnTBAP. An untreated control was included. The sperm viability, ROS production, total and progressive motility were evaluated. The results showed that the direct exposure of spermatozoa to MnTBAP significantly decreases the ROS levels in comparison with the untreated control without affecting the viability. The supplementation of the vitrification medium with MnTBAP did not affect the parameters analysed. However, the supplementation of the warming and incubation post-warming media resulted in a decrease in ROS production and maintained viability and motility for 4 hr after warming with concentrations up to 100 µM of MnTBAP. Higher concentrations of MnTBAP caused a decrease in total motility. In conclusion, the use of MnTBAP during the warming or post-warming incubation media has beneficial effect decreasing ROS levels and maintaining the viability and motility during the vitrification procedure.

The Effect of N-N-Dimethylformamide on the Membrane Characteristics of Canine Spermatozoa After Cryopreservation, and its Relationship With Post-Thaw Motility.

Some studies have demonstrated that glycerol is superior to amides in preserving sperm motion characteristics of canine sperm; however, little is known about the effect of these cryoprotectants on the membrane characteristics of canine spermatozoa after freezing/thawing. In this study, the effects of using either N-N-dimethylformamide (DMF) or glycerol (GLY) on the integrity and function of the canine sperm, after cryopreservation were determined. We hypothesized that the use of a multiparametric approach for assessing the effect of DMF on the membranes of canine sperm would explain the lower values reported for post-thaw motility. Ejaculates from 12 dogs were collected, split into 2 groups, and frozen using a tris-fructose-citrate-egg yolk-based extender containing either 7% (v/v) GLY or 7% (v/v) DMF. Frozen straws (n = 120) were thawed and analyzed for subjectively-assessed sperm progressive motility, normal morphology, plasma membrane integrity, plasma membrane function (HOST+), acrosome membrane integrity, high mitochondrial membrane potential, and simultaneous assessment of sperm membrane integrity and function by a triple-staining fluorescent procedure. Overall, sperm motility and membrane intactness/function were higher when GLY was used as a cryoprotectant, as compared to DMF (P < .05). A model to explain the variation in progressive motility using the values obtained from the sperm integrity and function parameters was designed. The percent HOST+ sperm and high mitochondrial membrane potential sperm were mostly associated with the changes observed in the progressive motility (r2 = 0.84; P = .043) when either GLY or DMF were used as cryoprotectants. These results may explain the overall reduced sperm quality observed after cryopreservation, as a reflection of sublethal damage sustained by the sperm membranes.

Equine Chorionic Gonadotropin (eCG) improves bucks' semen quality during the nonbreeding season.

In most goat breeds, testosterone serum concentration and semen quality decrease during the nonbreeding season. However, bucks reproductive activity may be stimulated with the administration of equine chorionic gonadotropin (eCG). Therefore, the aim of this study was to determine whether the repeated administration of eCG stimulates the reproductive status of bucks during the nonbreeding season. The study was performed with 19 bucks that were assigned to a group that was treated with eCG (GeCG) and an untreated control group (GCon). The GeCG bucks received an initial dose of 800 IU of eCG (Day 0), followed by four doses of 500 IU administered every 5 days beginning on Day 5. Serum testosterone and anti-eCG antibody concentrations, testicular and seminal traits were determined until Day 60. Testosterone concentration (from Day 3 to 21: p < 0.0001), anti-eCG titre (from Day 12 to 44: p ≤ 0.01), percentage of motile spermatozoa (Day 6: p = 0.006 and 14: p = 0.001) and of spermatozoa with progressive motility (Day 6: p = 0.01 and 14: p = 0.002) and the percentage of spermatozoa with functional membrane (Day 6: p = 0.02 and 22: p = 0.008) were higher in GeCG than in GCon bucks. Also in frozen-thawed samples, the percentage of motile spermatozoa tended to be higher in GeCG than that of GCon bucks (p = 0.07). In conclusion, the administration of eCG during the nonbreeding season stimulated the secretion of testosterone and improved fresh and possibly frozen-thawed semen quality. However, it also resulted in an increase in anti-eCG antibody titre.

Effect of incubation temperature after devitrification on quality parameters in human sperm cells.

Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 °C) and (ii) incubated at 37 °C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (ΔΨM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and ΔΨM compared to the control, while the incubation at 37 °C promoted a decrease in these parameters. The ROS levels were increased at both incubation conditions. The progressive motility was decreased in all experimental groups and the decrease was more pronounced under incubation at RT. No increase in phosphatidylserine externalization was observed. In conclusion, prior to use in assisted reproduction procedures, devitrified spermatozoa at RT conserve a better viability and ΔΨM than at 37 °C.

Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation / Inibidor do ativador do plasminogênio 1 e antipaína preservam a integridade do acrossoma de espermatozoides bovinos durante a criopreservação

Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.(AU)

A criopreservação é parcialmente prejudicial à fertilidade do sêmen de bovinos e induz mudanças semelhantes à capacitação em espermatozoides. O plasma seminal contém serina-proteases e inibidores de serina-proteases que estão envolvidos na fertilização de mamíferos, e os inibidores podem ser aplicados para evitar uma capacitação espermática induzida pelo frio. Analisaram-se os efeitos de diferentes concentrações de dois inibidores de serina-proteases, inibidor do ativador do plasminogênio 1 - PAI-1 (70ƞg, 140ƞg e 210ƞg) e antipaína (10µg, 50µg e 100µg) na suplementação ao diluidor de criopreservação de sêmen bovino. Trinta e seis ejaculados de quatro bovinos Curraleiro Pé-Duro foram usados para criopreservação. Os efeitos dos inibidores sobre os parâmetros dos espermatozoides (cinética espermática - CASA, integridade acrossomal, integridade da membrana plasmática, potencial de membrana mitocondrial, defeitos espermáticos e taxa de reação acrossomal) foram avaliados no sêmen pós-descongelamento. A criopreservação de espermatozoides com antipaína diminuiu os parâmetros cinéticos pós-descongelamento de MP, VSL, LIN, SRT e a porcentagem de espermatozoides hiperativados, PAI-1 (210ƞg) diminuiu VSL e LIN. Antipaína e PAI-1 não tiveram efeitos nos parâmetros de integridade da membrana plasmática, no potencial de membrana mitocondrial e nos defeitos espermáticos. Espermatozoides criopreservados na presença de antipaína e PAI-1 (70 e 140ƞg) preservaram a integridade acrossomal, assim como foram capazes de completar a reação acrossômica in vitro. Em conclusão, os inibidores de serina-proteases, antipaína e PAI-1 (70 e 140ƞg) são capazes de preservar a integridade acrossomal de espermatozoides criopreservados de bovinos.(AU)

Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation / Inibidor do ativador do plasminogênio 1 e antipaína preservam a integridade do acrossoma de espermatozoides bovinos durante a criopreservação

Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.(AU)

A criopreservação é parcialmente prejudicial à fertilidade do sêmen de bovinos e induz mudanças semelhantes à capacitação em espermatozoides. O plasma seminal contém serina-proteases e inibidores de serina-proteases que estão envolvidos na fertilização de mamíferos, e os inibidores podem ser aplicados para evitar uma capacitação espermática induzida pelo frio. Analisaram-se os efeitos de diferentes concentrações de dois inibidores de serina-proteases, inibidor do ativador do plasminogênio 1 - PAI-1 (70ƞg, 140ƞg e 210ƞg) e antipaína (10µg, 50µg e 100µg) na suplementação ao diluidor de criopreservação de sêmen bovino. Trinta e seis ejaculados de quatro bovinos Curraleiro Pé-Duro foram usados para criopreservação. Os efeitos dos inibidores sobre os parâmetros dos espermatozoides (cinética espermática - CASA, integridade acrossomal, integridade da membrana plasmática, potencial de membrana mitocondrial, defeitos espermáticos e taxa de reação acrossomal) foram avaliados no sêmen pós-descongelamento. A criopreservação de espermatozoides com antipaína diminuiu os parâmetros cinéticos pós-descongelamento de MP, VSL, LIN, SRT e a porcentagem de espermatozoides hiperativados, PAI-1 (210ƞg) diminuiu VSL e LIN. Antipaína e PAI-1 não tiveram efeitos nos parâmetros de integridade da membrana plasmática, no potencial de membrana mitocondrial e nos defeitos espermáticos. Espermatozoides criopreservados na presença de antipaína e PAI-1 (70 e 140ƞg) preservaram a integridade acrossomal, assim como foram capazes de completar a reação acrossômica in vitro. Em conclusão, os inibidores de serina-proteases, antipaína e PAI-1 (70 e 140ƞg) são capazes de preservar a integridade acrossomal de espermatozoides criopreservados de bovinos.(AU)

Semen quality before cryopreservation and after thawing in 543 patients with testicular cancer.

OBJECTIVE: The main objective of this study was to assess semen characteristics of patients with testicular cancer before cryopreservation and after thawing, to evaluate the consequences of this technique on sperm quality in patients with testicular cancer. METHODS: Five hundred eighty-nine samples from 543 patients with testicular cancer were cryopreserved between 1995 and 2015, one aliquot per patient was used for a thawing test to assess the impact of cryopreservation on sperm motility; semen analysis was performed before cryo preservation and after thawing, the result interpretation was carried out using the 2010 World Health Organization (WHO) Laboratory Manual, and consent forms were signed by the patients for freezing and when sperm was used for reproductive purposes. RESULTS: Hypospermia was observed in 28.7% of samples, the median sperm concentration was 18 million/mL with 35% oligozoospermia; twenty-two patients (4.1%) had azoospermia and 12.7% had severe oligozoospermia, the median sperm count was 31.3 million and 261 semen samples (44.3%) were normal in all parameters according to the WHO; total motile sperm count before cryopreservation and after thawing was 12 (0-412.2) and 7 (0-303.9) million sperm, respectively (p < 0.00001, 95% CI 5.48-14.91), which represents a 32% reduction; concerning the utilization of cryopreserved semen samples, only twelve patients (2.2%) used their frozen sperm for reproductive purposes. CONCLUSIONS: An impairment in semen quality was found in almost half of the samples from patients with testicular cancer, only few patients had azoospermia or severe oligozoospermia; sperm cryopreservation significantly reduces sperm motility and total motile sperm count and very few patients use their frozen sperm for reproductive purposes.

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm.

In addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.

Cryopreservation of Peruvian Paso horse spermatozoa: dimethylacetamide preserved an optimal sperm function compared to dimethyl sulfoxide, ethylene glycol and glycerol.

The objective was to evaluate the effect of different cryoprotectant agents in the cryopreservation of Peruvian Paso horse semen. Twenty semen samples were collected from five Peruvian Paso horse stallions. Each sample was divided into 12 parts to form the groups: dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol (GLY), at 3%, 4% and 5%. Samples were frozen using a rate-controlled freezer. Sperm parameters evaluated were motility and viability/acrosomal status. After thawing, progressive motility in DMA group was higher (p < .05) than in DMSO, EG and GLY groups. Similarly, viable acrosome-intact spermatozoa were higher (p < .05) using DMA in comparison with DMSO. No differences were found when comparing concentrations for any of the cryoprotectant agents. In conclusion, DMA seems to be a good cryoprotectant agent for the cryopreservation of Peruvian Paso horse stallion semen.

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs / Teste de ligação espermática à membrana perivitelínica da gema de ovo de galinha como avaliação funcional do sêmen de cães

During fertilization, spermatozoa interact with the zona pellucida (ZP) through the binding between the acrosome and proteins 2 and 3 (ZP2 and ZP3). The perivitelline membrane of chicken egg yolk is homologous to the mammalian ZP3, which allows the binding of sperm of several species. The aim of this study was to standardize and evaluate the efficiency of sperm-binding to the perivitelline membrane of chicken eggs as a functional method for canine semen evaluation. For this purpose, nine post-thaw sperm samples were used, which were divided into two aliquots: the first was kept in water bath at 37ºC (live sample) and the second was submitted to cold shock to induce cellular damage (dead sample). The two aliquots were mixed on five proportions, corresponding to 0%, 25%, 50%, 75%, and 100% of viable cells, and the binding test was performed by analyzing the number of spermatozoa bonded to the perivitelline membrane by means of computerized assessment of sperm motility (CASA) or conventional microscopy. Additionally, samples were submitted to sperm motility analysis, evaluation of plasmatic and acrosomal membrane integrity, and sperm mitochondrial activity. The sperm-binding test to the perivitelline membrane of chicken egg yolk was considered a feasible sperm analysis test for both fertilizing capacity and overall sperm attributes evaluation, mainly when the analysis is performed by a conventional microscope, which expands its practicality to the majority of canine reproduction laboratories.(AU)

Durante a fecundação, os espermatozoides interagem com a zona pelúcida (ZP) por meio da ligação entre o acrossomo e as proteínas 2 e 3 (ZP2 e ZP3). A membrana perivitelínica da gema de ovo de galinhas é homóloga à ZP3 de mamíferos, possibilitando a ligação espermática de diversas espécies. Este trabalho padronizou e avaliou a eficiência do teste de ligação espermática à membrana perivitelínica da gema de ovo de galinhas como avaliação funcional do sêmen de cães. Para tal, foram utilizadas nove amostras seminais previamente criopreservadas. Cada amostra foi dividida em duas alíquotas: a primeira foi mantida em banho-maria à 37ºC (vivos) e a segunda submetida a choque térmico com o intuito de induzir dano celular (mortos). As duas alíquotas foram misturadas, correspondendo a 0, 25, 50, 75 e 100% de células viáveis. As amostras foram avaliadas quanto ao número de espermatozoides ligados à membrana perivitelínica por meio da análise computadorizada da motilidade (CASA) ou microscopia convencional. Ademais, as amostras foram avaliadas quanto à motilidade espermática, integridade das membranas acrossomal e plasmática e atividade mitocondrial espermática. O teste de ligação espermática à membrana perivitelínica de ovos de galinha foi considerado um teste de análise seminal exequível tanto para avaliar a capacidade fecundante dos espermatozoides como atributos seminais gerais, especialmente quando realizado em microscopia convencional, expandindo sua praticidade para a maioria dos laboratórios de análise de sêmen canino.(AU)

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs / Teste de ligação espermática à membrana perivitelínica da gema de ovo de galinha como avaliação funcional do sêmen de cães

During fertilization, spermatozoa interact with the zona pellucida (ZP) through the binding between the acrosome and proteins 2 and 3 (ZP2 and ZP3). The perivitelline membrane of chicken egg yolk is homologous to the mammalian ZP3, which allows the binding of sperm of several species. The aim of this study was to standardize and evaluate the efficiency of sperm-binding to the perivitelline membrane of chicken eggs as a functional method for canine semen evaluation. For this purpose, nine post-thaw sperm samples were used, which were divided into two aliquots: the first was kept in water bath at 37ºC (live sample) and the second was submitted to cold shock to induce cellular damage (dead sample). The two aliquots were mixed on five proportions, corresponding to 0%, 25%, 50%, 75%, and 100% of viable cells, and the binding test was performed by analyzing the number of spermatozoa bonded to the perivitelline membrane by means of computerized assessment of sperm motility (CASA) or conventional microscopy. Additionally, samples were submitted to sperm motility analysis, evaluation of plasmatic and acrosomal membrane integrity, and sperm mitochondrial activity. The sperm-binding test to the perivitelline membrane of chicken egg yolk was considered a feasible sperm analysis test for both fertilizing capacity and overall sperm attributes evaluation, mainly when the analysis is performed by a conventional microscope, which expands its practicality to the majority of canine reproduction laboratories.(AU)

Durante a fecundação, os espermatozoides interagem com a zona pelúcida (ZP) por meio da ligação entre o acrossomo e as proteínas 2 e 3 (ZP2 e ZP3). A membrana perivitelínica da gema de ovo de galinhas é homóloga à ZP3 de mamíferos, possibilitando a ligação espermática de diversas espécies. Este trabalho padronizou e avaliou a eficiência do teste de ligação espermática à membrana perivitelínica da gema de ovo de galinhas como avaliação funcional do sêmen de cães. Para tal, foram utilizadas nove amostras seminais previamente criopreservadas. Cada amostra foi dividida em duas alíquotas: a primeira foi mantida em banho-maria à 37ºC (vivos) e a segunda submetida a choque térmico com o intuito de induzir dano celular (mortos). As duas alíquotas foram misturadas, correspondendo a 0, 25, 50, 75 e 100% de células viáveis. As amostras foram avaliadas quanto ao número de espermatozoides ligados à membrana perivitelínica por meio da análise computadorizada da motilidade (CASA) ou microscopia convencional. Ademais, as amostras foram avaliadas quanto à motilidade espermática, integridade das membranas acrossomal e plasmática e atividade mitocondrial espermática. O teste de ligação espermática à membrana perivitelínica de ovos de galinha foi considerado um teste de análise seminal exequível tanto para avaliar a capacidade fecundante dos espermatozoides como atributos seminais gerais, especialmente quando realizado em microscopia convencional, expandindo sua praticidade para a maioria dos laboratórios de análise de sêmen canino.(AU)

Effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine on membrane lipid profile and cryotolerance of human sperm.

OBJECTIVE: To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters. DESIGN: Experimental study. SETTING: University-affiliated tertiary hospital. PATIENT(S): A total of 20 normospermic fertile men. INTERVENTION(S): Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC). MAIN OUTCOME MEASURE(S): Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). RESULT(S): Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC. CONCLUSION(S): Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC.