Effects of antioxidant Bis-carboxyethyl germanium sesquioxide added to bovine semen cryopreservation medium on in vitro assessed morphofunctional sperm parameters.

This study investigated the impact of various Ge132 (Bis-carboxyethyl germanium sesquioxide) concentrations on frozen bovine semen. Ejaculates from three bulls were pooled and divided into six groups, each one with different Ge132 concentrations (0, 500, and 1000 µg/mL) and each group was incubated in different conditions (33°C for 30 min (D: D0, D500, and D1000), and the other was immediately cooled to 4°C (R: R0-control; R500 and R1000)). Thawed semen was evaluated for sperm characteristics by CASA and flow cytometer. Results showed better motility in the immediate cooling group without Ge132 compared with high Ge132 concentrations. Values for total motility dropped after 5 and 60 min in groups with high Ge132 levels and some control groups. Linearity increased with 1000 µg/mL Ge132, while straightness differed between moments in multiple groups. Membrane integrity was higher in a control group and certain Ge132 groups. Lower O2 - generation occurred without Ge132. After oxidative stress induction, lipid peroxidation intensity increased with arachidonic acid, but D1000 had lower peroxidation than R0. Overall, Ge132 appears to have provided protection against PLM when subjected to oxidative stress, since even at high concentrations it maintained sperm metabolism.

In vitro exposure of porcine spermatozoa to methylparaben, and propylparaben, alone or in combination adversely affects sperm quality.

Parabens (PBs) are widely used in the cosmetic, pharmaceutical, and food industries as preservatives of products. Because of its great use, humans and other organisms are highly exposed daily. However, little is known about the effect of PBs on male infertility. Therefore, the aim of the present study was to evaluate the effect of methylparaben (MePB) and propylparaben (PrPB), alone or in combination, on the physiological characteristics of pig in vitro exposed sperm to different concentrations (0, 200, 500, and 700 µM) for viability, motility, and acrosome integrity evaluation and (0, 200, 500, 700, 1000, and 2000 µM) for DNA fragmentation index evaluation, after 4 h of exposure. The results showed that sperm viability decreased after exposure to MePB from the concentration of 500 µM. In the PrPB and mixture groups, viability decreased at all concentrations except for the control. The decrease in viability of sperm exposed to PrPB was greater than that of the mixture and MePB groups. Sperm motility decreased in all the experimental groups exposed to PBs, at all concentrations, except for the control group. Acrosome integrity was not decreased in the MePB group; however, in the PrPB group, it decreased at a concentration of 200 µM and in the mixture at 500 µM. All groups exhibited DNA damage at different concentrations, except for the control group. Additionally, the effect of PBs on sperm quality was concentration-dependent. The results demonstrated that MePB and PrPB alone or in combination can have adverse effects on sperm quality parameters. MePB had lower toxicity than did both PrPB and the mixture. The mixture did not have an additive effect on any of the parameters evaluated. This could partially explain the link between PB exposure and infertility.

Effect of Three Semen Extenders on Sperm Quality and In Vitro Fertilization Rates of Fresh and Cryopreserved Sperm Collected from Llama (Lama glama) Vas Deferens.

The advances in Assisted Reproductive Technologies (ARTs) applied in South American camelid species are still scarce. The aim of this study was to compare the effects of three semen extenders, before and after the cryopreservation of spermatozoa obtained from the vas deferens, on sperm quality parameters and in vitro fertilization rates of llama (Lama glama) oocytes. Mature fertile llama males (Lama glama; n = 6; age: 48-60 mo.; BCS: ~2.7) were included in the study. Sperm samples were collected from each male using the surgical technique of the vas deferens deviation. Then, the sperm samples were pooled and diluted with the Tris-EY, Andromed®, or BioxCell® extender in order to subsequently carry out the sperm cryopreservation process. The sperm quality assessment related to each extender was performed before and after cryopreservation with regard to sperm morphological abnormalities, acrosome integrity, sperm viability, membrane permeability, and sperm motility traits. Moreover, in vitro fertilization (IVF) procedures were carried out to evaluate the in vitro fertility of the cryopreserved sperm samples using each extender. Overall, significant differences were observed before and after cryopreservation regarding acrosome integrity, sperm viability, membrane permeability, and sperm motility traits among the extenders used, where Tris-EY and Andromed® were better than BioxCell® (p < 0.05); however, no differences were observed regarding the sperm morphological abnormalities among extenders (p > 0.05). Moreover, multiple differences were observed with regard to the velocity and linearity kinematic parameters obtained by computerized analysis before and after the cryopreservation process, irrespective of the extender used (p < 0.05). Finally, differences were observed regarding the in vitro fertilization rates among the different extender-derived samples (p < 0.05). In conclusion, the sperm quality using Tris-EY and Andromed® was better before and after cryopreservation compared to that using BioxCell®. Although the number of fertilized oocytes obtained after the IVF process between Tris-EY and Andromed® was similar, Andromed®-derived samples showed the best sperm quality results before and after cryopreservation. This indicates that the cryopreservation extender is a determining factor in significantly improving in vitro fertilization rates when using sperm samples obtained from vas deferens in llama (Lama glama) males.

Effect of Arthrospira (Spirulina) maxima on Cadmium-Chloride-Induced Alterations in Sexual Behavior and Fertility in Male Wistar Rats.

Chronic exposure to potentially toxic elements (PTEs) such as cadmium (Cd) leads to male reproductive toxicity through the generation of oxidative stress. Spirulina Arthrospira maxima (AM) is a cyanobacterium that has been consumed since ancient times for its high nutritional value, and in recent years for its antiviral, hepatoprotective, hypoglycemic, anticancer, and antioxidant effects, among others. This study aimed to evaluate the effects of AM against the damage to reproductive health induced by Cd. A total of 48 10-week-old sexually experienced male Wistar rats were distributed in five groups (n = 8): control; vehicle (tween-water); cadmium chloride (CdCl2) 5 mg/kg; and three doses of AM (100, 200 and 400 mg/kg) + CdCl2 5 mg/kg. All treatments were orally administered once a day for 36 consecutive days. At the end, sexual behavior was evaluated, and semen, testicle, and blood samples were obtained to analyze sperm quality, enzymatic activity, and testosterone levels, respectively. Rats exposed to Cd showed a decrease in sexual behavior, as well as in the quality of reproductive health, and an increase in oxidative stress; while rats exposed simultaneously to AM + Cd showed an improvement in all this parameters. Based on our results, we believe that the mechanism by which AM exerts its effect could be attributed to the presence of phycobiliproteins. These compounds are responsible for exerting an antioxidant effect and chelating effect on elements such as Cd.

Effects of major analgesics on male fertility: A systematic literature review.

OBJECTIVE: To verify, based on a systematic literature review, the effects of the main analgesics on male fertility. DATA SOURCES: The studies were analyzed from the PubMed, SciELO and LILACS databases. STUDY SELECTION: The articles selected for the present review included: cohort studies; cross-sectional studies, clinical trials; complete studies; studies with animal models that addressed the proposed theme and that were published within the stipulated period from March 1, 2013, to March 31, 2023, in English, Portuguese and Spanish. These would later have to go through inclusion stages such as framing the type of study and exclusion criteria. DATA COLLECTION: Author's name, year of publication, study population, number of patients, analgesic, administration time, dose, and effect. CONCLUSIONS: There are in vitro and in vivo studies that link paracetamol and ibuprofen to endocrine and seminal changes that are harmful to male fertility. However, more clinical research is needed to determine the doses and timing of administration that affect fertility. The effects of aspirin on male fertility are still unclear due to the lack of studies and consistent methodologies. There is not enough research on dipyrone and its relationship with male fertility, requiring more studies in this area.

A systematic review focused on lubricant use and sperm quality: Improving human reproductive success by informing lubricants toxicity.

Lubricants play a pivotal role in human reproductive health, particularly concerning their impact on sperm parameters. In this systematic review, we assess the implications of both synthetic and natural or organic lubricants on sperm health and fertility, based on a compilation of 20 distinct studies. Synthetic lubricants, including K-Y Jelly, Replens, and Astroglide, predominantly containing ingredients like methylparaben and glycerin, have been linked to detrimental effects on sperm motility and chromatin integrity. Chemical characteristics, notably osmolality and pH, are central to understanding these effects. Despite the World Health Organization's osmolality recommendation of 380 mOsm/kg, many commercial products surpass this. Natural solutions offer varied results, while olive oil exhibits unfavorable effects on sperm health, egg white proves non-toxic, potentially benefitting sperm health. Conversely, Pre-Seed, widely endorsed in the research community, generally demonstrates minimal adverse impact on sperm. The review highlights the significance of lubricant selection in evidence-based reproductive strategies.

Cryopreservation-Induced Morphological Changes in Freshwater Fish Sperm: A Systematic Review.

A systematic review was performed to summarize the scientific evidence and critically evaluate the effects of cryopreservation on sperm morphology in freshwater fish, and to assess the methodologies for sperm morphology classification. The search strategy was applied to four electronic databases (CAB Direct, Pub Med, Scopus, and ISI Web of Science). The main inclusion criteria involved studies on semen from freshwater fish subjected to the cryopreservation process and evaluation of sperm quality through morphology. The risk of bias was assessed with respect to randomization, allocation concealment, blinding, incomplete outcome data, and selective reporting. A total of 6 publications reporting sperm cryopreservation from 4 species with a total 74 fish individuals were included in this review. A high methodological variability among the results of the studies was observed due to the species-specific protocols and diversity of freshwater fish species studied. All included studies reported negative effects of cryopreservation on sperm quality, especially morphology, highlighting the increase in incidence of sperm abnormalities. However, only five studies statistically compared abnormalities between groups (fresh and cryopreserved sperm). Our results suggest the need to elaborate on a new morphological classification of fish spermatozoa, by considering the structure and physiology of fish sperm. This classification should be developed based on the sperm characterization and observing damage caused by different cryopreservation protocols.

Persistent oligonecrozoospermia after asymptomatic SARS-CoV-2 infection. A case report and literature review.

COVID-19 is known to have deleterious effects on different systems such as the respiratory, cardiovascular, central nervous, and gastrointestinal. However, conflicting data about the possible implications for male reproductive health and fertility have been reported. In addition, the long-term consequences of SARS-CoV-2 infection remain unclear. Herein, we report a case of a 42-year-old man with no known co-morbidities and normal baseline semen quality, who subsequently suffered an asymptomatic SARS-CoV-2 infection. Shortly after, the patient developed sudden oligoasthenozoospermia, even reaching azoospermia, which gradually evolved into persistent severe oligonecrozoospermia, accompanied by semen inflammation and oxidative stress. Remarkably, the latter occurred in the absence of urogenital infections, hormonal imbalances, tissue/organ obstruction/damage, medication or drug treatment, smoking, or exposure to toxins/pollutants, radiation, or high temperature. This case constitutes valuable clinical evidence that adds to the current knowledge in the field and highlights the need for further and longer follow-up studies to better understand the putative long-term consequences of SARS-CoV-2 infection on male fertility.

Ram semen quality after supplementation with gelatin, agar or alginate prior to cooling storage.

Adding gelling agents to convert the liquid state of the semen extender to a solid state allows for an increased sperm life span. Gelatin and alginate have been used to study the effects of gelling agents on sperm quality. However, there are other gelling agents that have not been studied, such as agar. In addition, studying different sources of gelling agents or the effect of mixing more than one gelling agent with semen extenders on sperm fertility has received little attention. Therefore, the objective of this study was to evaluate the effect of adding agar and a mixture of gelling agents from different sources to semen extender on ram sperm traits and fertility. The first trial evaluated the effect of the addition of 2.5-3 mg mL-1 of gelatin mixed with 0.5-20 mg mL-1 of agar or alginate to ram semen extender on sperm (motility, progressive motility, live/dead, membrane integrity) and semen (pH) characteristics. The response variables were evaluated 1, 72 and 144 h after storage at 4°C. In the second trial, two sources (feed grade and bacteriological) of gelatin and agar were evaluated on the response variables as in Trial 1. In trial 3, a total of 34 ewes were inseminated with doses supplemented (n = 17) with or without (n = 17) agar and gelatin. The pregnancy rate was diagnosed 40 days after insemination. In general, adding agar and gelatin improves (p < .05) sperm motility, membrane integrity and the ratio of live sperm after 144 h of storage compared to the Control group, regardless of the source (bacteriological or feed grade). However, the pregnancy rate in ewes was not influenced (p ≥ .05) by semen doses stored with agar and gelatin. In conclusion, the addition of agar and gelatin preserves ram sperm motility and membrane integrity after 144 of storage at 4°C without affecting the pregnancy rate in inseminated ewes.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) alleviate paclitaxel-induced spermatogenesis defects and maintain male fertility.

Chemotherapeutic drugs can cause reproductive damage by affecting sperm quality and other aspects of male fertility. Stem cells are thought to alleviate the damage caused by chemotherapy drugs and to play roles in reproductive protection and treatment. This study aimed to explore the effects of human umbilical cord mesenchymal stem cells (hUC-MSCs) on alleviating paclitaxel (PTX)-induced spermatogenesis and male fertility defects. An in vivo PTX-induced mice model was constructed to evaluate the reproductive toxicity and protective roles of hUC-MSCs in male fertility improvement. A 14 day PTX treatment regimen significantly attenuated mice spermatogenesis and sperm quality, including affecting spermatogenesis, reducing sperm counts, and decreasing sperm motility. hUC-MSCs treatment could significantly improve sperm functional indicators. Mating experiments with normal female mice and examination of embryo development at 7.5 days post-coitum (dpc) showed that hUC-MSCs restored male mouse fertility that was reduced by PTX. In IVF experiments, PTX impaired sperm fertility and blastocyst development, but hUC-MSCs treatment rescued these indicators. hUC-MSCs' protective role was also displayed through the increased expression of the fertility-related proteins HSPA2 and HSPA4L in testes with decreased expression in the PTX-treated group. These changes might be related to the PTX-induced decreases in expression of the germ cell proliferation protein PCNA and the meiosis proteins SYCP3, MLH1, and STRA8, which were restored after hUC-MSCs treatment. In the PTX-treated group, the expression of testicular antioxidant proteins SIRT1, NRF2, CAT, SOD1, and PRDX6 was significantly decreased, but hUC-MSCs could maintain these expressions and reverse PTX-related increases in BAX/BCL2 ratios. hUC-MSCs may be a promising agent with antioxidant and anti-apoptosis characteristics that can maintain sperm quality following chemotherapy treatment.

COVID-19 associates with semen inflammation and sperm quality impairment that reverses in the short term after disease recovery.

Introduction: COVID-19 exerts deleterious effects on the respiratory, cardiovascular, gastrointestinal, and central nervous systems, causing more severe disease in men than in women. However, cumulative reported data about the putative consequences on the male reproductive tract and fertility are controversial. Furthermore, the long-term effects of SARS-CoV-2 infection are still uncertain. Methods: In this study, we prospectively evaluated levels of inflammatory cytokines and leukocytes in semen and sperm quality parameters in a cohort of 231 reproductive-aged male patients, unvaccinated, who had recovered from mild or severe COVID-19 and in 62 healthy control individuals. Sperm quality was assessed early (less than 3 months) and long (more than 3 and up to 6 months) after having COVID-19. Interestingly, and unlike most reported studies, available extensive background and baseline data on patients' sperm quality allowed performing a more accurate analysis of COVID-19 effects on sperm quality. Results: Significantly higher levels of IL-1ß, TNF and IFNγ were detected in semen from patients recently recovered from mild and/or severe COVID-19 with respect to control individuals indicating semen inflammation. Moreover, patients recovered from mild and/or severe COVID-19 showed significantly reduced semen volume, lower total sperm counts, and impaired sperm motility and viability. Interestingly, all observed alterations returned to baseline values after 3 or more months after disease recovery. Discussion: These results indicate that COVID-19 associates with semen inflammation and impaired semen quality early after disease. However, long COVID-19 seems not to include long-term detrimental consequences on male fertility potential since the observed alterations were reversible after 1-2 spermatogenesis cycles. These data constitute compelling evidence allowing a better understanding of COVID-19 associated sequelae, fundamental for semen collection in assisted reproduction.

Mammals' sperm microbiome: current knowledge, challenges, and perspectives on metagenomics of seminal samples.

Bacterial growth is highly detrimental to sperm quality and functionality. However, during the last few years, using sequencing techniques with a metagenomic approach, it has been possible to deepen the study of bacteria-sperm relationships and describe non-culturable species and synergistic and antagonistic relationships between the different species in mammalian animals. We compile the recent metagenomics studies performed on mammalian semen samples and provide updated evidence to understand the importance of the microbial communities in the results of sperm quality and sperm functionality of males, looking for future perspectives on how these technologies can collaborate in the development of andrological knowledge.

Morin improves Bisphenol-A-induced toxicity in the rat testicular mitochondria and sperms.

OBJECTIVE: The present study aimed to examine the ameliorative effects of Morin (MRN) on Bisphenol-A (BPA)-induced oxidative stress in testicular mitochondria and sperm quality of rats. METHODS: BPA and MRN (25, 50, and 100 µM) were given to the spermatozoa and testicular tissue mitochondria. The sperm quality, mitochondrial viability, and MMP (mitochondrial membrane potential) were examined. Superoxide dismutase, CAT (catalase), malondialdehyde, and reactive oxygen species (ROS) levels of rat testicular mitochondria were measured. RESULTS: BPA raised mitochondrial oxidative stress biomarkers, whereas antioxidant acclivity and MMP were significantly lowered. BPA significantly lowered the normality, viability, and motility of the sperms. MRN dose-dependently lowered oxidative stress of the mitochondria, raised MMP, as well as improved the percentage of abnormality, motility, and viability of the sperms. CONCLUSIONS: These data demonstrated that MRN dose-dependently attenuated BPA-induced mitochondrial damage and improved sperm quality by preventing oxidative stress.

Melatonin Supplementation for the Cryopreservation of Canine Sperm.

Antioxidants can be used in sperm cryopreservation protocols to reduce oxidative stress that occurs due to the cryopreservation process. The aim of this study was to evaluate the effects of melatonin supplementation on quality and oxidative stress parameters in cryopreserved canine sperm. Eighteen sperm ejaculates were collected from 6 Frenchie Bulldog males (3 collections per male). Sperm motility parameters, membrane integrity, and sperm morphology were analyzed before the cryopreservation process. The extender used in cryopreservation was composed of Tris-egg yolk and ethylene glycol 5% was added as a cryoprotectant. The cryoprotective medium was supplemented with 1.0, 1.5, 2.0, 2.5, and 3.0 mM melatonin, and the control group (without melatonin). Post-thaw sperm was evaluated as described for fresh sperm and oxidative stress parameters (lipid peroxidation, catalase, and superoxide dismutase). Post-thaw sperm motility parameters, membrane integrity, sperm morphology, and oxidative stress parameters did not differ (p > 0.05) among the control group and samples supplemented with melatonin. The results of this study showed that melatonin supplementation had no positive or negative effect on the parameters evaluated. Thus, it is suggested that different concentrations of melatonin be tested to assess its effectiveness as an antioxidant in the cryopreservation process in canine sperm.

Administration of carbetocin-a long-acting oxytocin analogue-before sperm collection by transrectal ultrasound-guided massage of the accessory sex glands in bucks (Capra hircus) and ibexes (Capra pyrenaica).

Transrectal ultrasonic-guided massage of the accessory sex glands (TUMASG) is a technique that allows collecting semen requiring few electrical stimuli or even no pulse. A long-acting analogue of oxytocin (carbetocin, 0.1 mg) was i.v. administered before TUMASG in 10 conscious bucks (Experiment 1) and 10 anaesthetized Iberian ibexes (Experiment 2) to shorten the time of semen collection, decrease the number of electrical stimuli and/or improve the semen quality. The ejaculated volume, concentration, quality parameters and kinetics variables of the sperm were determined in fresh semen. The time length of the procedures and the number of electric pulses applied were recorded. Furthermore, stress response indicators (number of vocalizations in Experiment 1; heart and respiratory rates, rectal temperature, cortisol levels, totals proteins and neutrophil-to-lymphocyte ratio in Experiment 2) were documented. In bucks, the administration of carbetocin tended to shorten the time needed for semen collection but no-showed differences in the fresh seminal quality. In the Iberian ibexes, there were no significant differences between groups in the time length of procedures or in the number of animals that ejaculated. Carbetocin administration only reduced the respiratory rate, did it modify fresh semen characteristics in ibexes. In conclusion, the administration of carbetocin did not appear as a useful tool to improve welfare during semen collection with TUMASG or semen quality in conscious bucks and anaesthetized ibexes, having only slight advantages related to the procedure.

Could parental high-fat intake program the reproductive health of male offspring? A review.

High-fat diet (HFD) intake can cause overweight and obesity and has become a global public health concern in recent years. Nutritional adversity at vulnerable windows of development can affect developing cells and their functions, including germ cells. Evidence shows that parental HFD intake prior to conception and/or during gestation and lactation could program the reproductive health of male offspring, ultimately resulting in impairment of the first as well as subsequent generations. In male offspring, adipose tissue and hypothalamic-pituitary-gonadal axis imbalance can impair the production of gonadotropins, leading to dysfunction of testosterone production and pubertal onset. The gonads can be directly impaired through oxidative stress, causing poor testosterone production and spermatogenesis; low sperm count, viability, and motility; and abnormal sperm morphology, which results in low sperm quality. Parental HFD intake could also be a risk factor for prostate hyperplasia and cancer in advanced age. It can impact the reproductive pattern of male offspring resulting in impairments in the subsequent generations. The investigation of semen quality must be extended to epidemiological and clinical studies of the male offspring of overweight and/or obese parents in order to improve the quality of human semen. This review addresses the effects of parental HFD intake on the reproductive parameters of male offspring and discusses the possible underlying mechanisms.

Effect of chronic sleep deprivation on acrosomal integrity and functional parameters of murine sperm.

OBJECTIVE: To evaluate the effect of chronic sleep deprivation on sperm function quality in mice. DESIGN: Experimental study. SETTING: Not applicable. ANIMALS: Spermatozoa from twenty-four 10-week-old C57BL/6J male mice. INTERVENTION(S): The sleep deprivation group underwent gentle handling for 6 hours for 5 consecutive days. The mice in the sleep recovery group were allowed to sleep during the 24-hour period after the sleep deprivation protocol. MAIN OUTCOME MEASURE(S): After euthanasia, the spermatozoa were collected for analysis. Sperm motility was evaluated using computer-assisted sperm analyzer. Intracellular superoxide anion (O2-) activity, acrosome integrity, mitochondrial activity, and DNA fragmentation assays were conducted afterward. RESULT(S): Sleep deprivation and sleep recovery groups presented a lower percentage of spermatozoa with an intact acrosome, compared with the respective control groups. Regarding DNA fragmentation, a decreased proportion of spermatozoa with Comet I class intact DNA was observed in the sleep recovery group, compared with the recovery control group. Beat cross frequency was increased in the sleep recovery group. CONCLUSION(S): Sleep deprivation can reduce sperm quality, impairing acrosome integrity. Sleep recovery decreased DNA integrity and increased beat cross frequency.

Dimethyl acetamide and dimethyl sulfoxide associated at glucose and egg yolk for cryopreservation of Pseudoplatystoma corruscans semen

This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility > 80%. After thawing, all treatments provided semen with total sperm motility > 40%, with no significant difference (P < 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P. corruscans semen samples.(AU)

Este estudo teve como objetivo desenvolver um protocolo para a criopreservação do sêmen de Pseudoplatystoma corruscans. Para tal, machos maduros foram induzidos hormonalmente com uma dose única de extrato de hipófise de carpa (5 mg/kg de peso vivo). O sêmen foi coletado e avaliado. Sendo testados para compor os diluentes, dois crioprotetores: dimetil acetamida (DMA) e dimetil sulfóxido (Me2SO), em duas concentrações (8% e 10%), + 5,0% glicose + 10% gema de ovo. O sêmen foi diluído na proporção 1: 4 (sêmen: extensor), embalado em palhetas de 0,5 mL e congelado em container dryshipper em vapores de nitrogênio líquido. Após o descongelamento, foram avaliados os aspectos cinéticos espermáticos, a morfologia espermática e a integridade do DNA dos espermatozoides criopreservados. Os machos de P. corruscans produziram sêmen com motilidade espermática > 80%. Todos os tratamentos proporcionaram após o descongelamento sêmen com motilidade espermática total > 40%, sem diferença significativa (P < 0,05) entre eles, como também entre os demais parâmetros cinéticos espermáticos avaliados. Os tratamentos com DMA proporcionaram uma menor fragmentação do DNA dos gametas. Malformações espermáticas foram identificadas, tanto no sêmen fresco, como no criopreservado, sendo identificado um aumento discreto dessas malformações nos espermatozoides das amostras de sêmen descongeladas de P. corruscans.(AU)

Influence of thawing temperature on sperm motility, structure, and metabolism of frozen bovine semen / Influência da temperatura de descongelamento na motilidade, estrutura e metabolismo do sêmen bovino congelado

ABSTRACT: The heating rate used during semen thawing plays an important role in reducing structural and functional damage to spermatozoa. In this study, we evaluated the influence of thawing temperature on semen quality, reactive oxygen species (ROS) production, and mitochondrial activity of cryopreserved bovine semen. A total of 195 straws of 0.5 mL from five Holstein Friesian bulls were used (39 straws per bull). Samples underwent 8 to 22 years of storage; they were processed under a standard protocol with tris-egg yolk and stored in liquid nitrogen. Samples were thawed for 30 seconds in a water bath at T1: 36 °C, T2: 38 °C or T3: 40 °C. Sperm motility and kinematics, morphology, structural membrane integrity (SMI), functional membrane integrity (FMI), acrosome integrity (AI), ROS, and mitochondrial membrane potential (ΔΨM) of post-thawing bovine sperm were evaluated. Generalized linear models were fitted to the data. Each model included the effects of bull, storage time, and treatment. The Shapiro-Wilk test was used to assess data normality, and means were compared using the Tukey test. T2 and T3 showed better results for sperm motility and kinematic parameters, SMI (%) (T1 41.9 ± 2.3; T2 45.7 ± 1.9; T3 47.4 ± 2.8), ROS (RFU/min) (T1 0.026 ± 0.007; T2 0.032 ± 0.001; T3 0.031 ± 0.001) and high-ΔΨM (RFU x 103) (67.1± 0,4; 71.3 ± 0.4; 74.2 ± 0.4) (P < 0.05). However, T1 had higher FMI (39.3 ± 2.3) than T2 (34.0 ± 1.9) (P < 0.05), though not significantly (P > 0.05) different from T3 (38.4 ± 2.2). Thawing temperatures of 38 °C and 40 °C increases motility, kinetics, membrane integrity, mitochondrial activity and ROS of cryopreserved bovine semen, compared with more conventional thawing at 36 °C.

RESUMO: A taxa de aquecimento usada durante o descongelamento do sêmen desempenha um papel importante na redução dos danos estruturais e funcionais nos espermatozóides. O objetivo desta pesquisa foi avaliar a influência da temperatura de descongelamento na qualidade do sêmen, produção de espécies reativas de oxigênio (ROS) e atividade mitocondrial do sêmen bovino criopreservado. Foram utilizados 195 palhetas de 0,5 mL de cinco touros Holstein Friesian (39 palhetas por touro). As amostras passaram por oito a 22 anos de armazenamento e foram processadas sob protocolo padrão com Tris-gema de ovo e armazenadas em nitrogênio líquido. As temperaturas de descongelamento foram T1: 36 °C, T2: 38 °C, T3: 40 °C, cada uma por 30 segundos em banho-maria. Pós-descongelamento, a motilidade e cinética dos espermatozoides, morfologia, integridade estrutural da membrana (SMI), integridade funcional da membrana (FMI), integridade acrossomal (AI), ROS e potencial de membrana mitocondrial (ΔΨM) foram avaliados. Modelos lineares generalizados foram ajustados. Cada modelo incluiu os efeitos de touro, tempo de armazenamento e tratamento. A normalidade dos dados foi avaliada pelo teste de Shapiro-Wilk e as médias comparadas pelo teste de Tukey. T2 e T3 apresentaram resultados mais elevados para a maioria dos parâmetros de motilidade e cinemática espermática, SMI (%) (T1 41,9 ± 2,3; T2 45,7 ± 1,9; T3 47,4 ± 2,8), ROS (RFU/min) (T1 0,026 ± 0,007; T2 0,032 ± 0,001; T3 0,031 ± 0,001) e alto ΔΨM (RFU x 103) (67,1 ± 0,4; 71,3 ± 0,4; 74,2 ± 0,4) (P < 0,05). No entanto, T1 apresentou maior FMI (%) (39,3 ± 2,3) em comparação a T2 (34,0 ± 1,9) (P < 0,05), mas não foi diferente do T3 (38,4 ± 2,2) (P > 0,05). Conclui-se que as temperaturas de descongelamento de 38 °C e 40 °C produzem um aumento na motilidade, cinética, integridade de membrana, atividade mitocondrial e ROS do sêmen bovino criopreservado, em comparação com o uso mais convencional de uma temperatura de descongelamento de 36 °C.

Influence of thawing temperature on sperm motility, structure, and metabolism of frozen bovine semen / Influência da temperatura de descongelamento na motilidade, estrutura e metabolismo do sêmen bovino congelado

The heating rate used during semen thawing plays an important role in reducing structural and functional damage to spermatozoa. In this study, we evaluated the influence of thawing temperature on semen quality, reactive oxygen species (ROS) production, and mitochondrial activity of cryopreserved bovine semen. A total of 195 straws of 0.5 mL from five Holstein Friesian bulls were used (39 straws per bull). Samples underwent 8 to 22 years of storage; they were processed under a standard protocol with tris-egg yolk and stored in liquid nitrogen. Samples were thawed for 30 seconds in a water bath at T1: 36 °C, T2: 38 °C or T3: 40 °C. Sperm motility and kinematics, morphology, structural membrane integrity (SMI), functional membrane integrity (FMI), acrosome integrity (AI), ROS, and mitochondrial membrane potential (ΔΨM) of post-thawing bovine sperm were evaluated. Generalized linear models were fitted to the data. Each model included the effects of bull, storage time, and treatment. The Shapiro-Wilk test was used to assess data normality, and means were compared using the Tukey test. T2 and T3 showed better results for sperm motility and kinematic parameters, SMI (%) (T1 41.9 ± 2.3; T2 45.7 ± 1.9; T3 47.4 ± 2.8), ROS (RFU/min) (T1 0.026 ± 0.007; T2 0.032 ± 0.001; T3 0.031 ± 0.001) and high-ΔΨM (RFU x 103) (67.1± 0,4; 71.3 ± 0.4; 74.2 ± 0.4) (P < 0.05). However, T1 had higher FMI (39.3 ± 2.3) than T2 (34.0 ± 1.9) (P < 0.05), though not significantly (P > 0.05) different from T3 (38.4 ± 2.2). Thawing temperatures of 38 °C and 40 °C increases motility, kinetics, membrane integrity, mitochondrial activity and ROS of cryopreserved bovine semen, compared with more conventional thawing at 36 °C.

A taxa de aquecimento usada durante o descongelamento do sêmen desempenha um papel importante na redução dos danos estruturais e funcionais nos espermatozóides. O objetivo desta pesquisa foi avaliar a influência da temperatura de descongelamento na qualidade do sêmen, produção de espécies reativas de oxigênio (ROS) e atividade mitocondrial do sêmen bovino criopreservado. Foram utilizados 195 palhetas de 0,5 mL de cinco touros Holstein Friesian (39 palhetas por touro). As amostras passaram por oito a 22 anos de armazenamento e foram processadas sob protocolo padrão com Tris-gema de ovo e armazenadas em nitrogênio líquido. As temperaturas de descongelamento foram T1: 36 °C, T2: 38 °C, T3: 40 °C, cada uma por 30 segundos em banho-maria. Pós-descongelamento, a motilidade e cinética dos espermatozoides, morfologia, integridade estrutural da membrana (SMI), integridade funcional da membrana (FMI), integridade acrossomal (AI), ROS e potencial de membrana mitocondrial (ΔΨM) foram avaliados. Modelos lineares generalizados foram ajustados. Cada modelo incluiu os efeitos de touro, tempo de armazenamento e tratamento. A normalidade dos dados foi avaliada pelo teste de Shapiro-Wilk e as médias comparadas pelo teste de Tukey. T2 e T3 apresentaram resultados mais elevados para a maioria dos parâmetros de motilidade e cinemática espermática, SMI (%) (T1 41,9 ± 2,3; T2 45,7 ± 1,9; T3 47,4 ± 2,8), ROS (RFU/min) (T1 0,026 ± 0,007; T2 0,032 ± 0,001; T3 0,031 ± 0,001) e alto ΔΨM (RFU x 103) (67,1 ± 0,4; 71,3 ± 0,4; 74,2 ± 0,4) (P < 0,05). No entanto, T1 apresentou maior FMI (%) (39,3 ± 2,3) em comparação a T2 (34,0 ± 1,9) (P < 0,05), mas não foi diferente do T3 (38,4 ± 2,2) (P > 0,05). Conclui-se que as temperaturas de descongelamento de 38 °C e 40 °C produzem um aumento na motilidade, cinética, integridade de membrana, atividade mitocondrial e ROS do sêmen bovino criopreservado, em comparação com o uso mais convencional de uma temperatura de descongelamento de 36 °C.