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Background Objectives Materials and Methods Results Discussion Conclusion Sperm cryopreservation is a consolidate option for long‐term male fertility preservation. The freezing/thawing procedure causes detrimental effects to spermatozoa, including damage to viability, motility, membrane composition, and DNA, whereas the effect on sperm chromatin compaction is less studied.The primary aim of this study was to investigate the impact of cryopreservation on sperm chromatin compaction. Furthermore, the effect of cryopreservation on sperm parameters (motility, viability, chromatin compaction, and DNA fragmentation) was also assessed in relation to the storage time in liquid nitrogen.Semen samples, collected from 126 (92 normozoospermic and 34 oligozoospermic) patients undergoing routine semen analysis in the Andrology Laboratory of Careggi University Hospital of Florence, were frozen by conventional fast vapor freezing method. Sperm motility, viability, kinematic parameters (by computer‐aided sperm analysis [CASA]), chromatin compaction (by staining with both aniline blue [AB] and Chromomycin A3 [CMA3]), and sperm DNA fragmentation (sDF, by TUNEL/Propidium Iodide [PI]) were evaluated before freezing and after thawing at different timepoints.After 7 days of storage, a significant decline in sperm motility, viability, and kinematics parameters, as well as a significant increase in the percentage of sperm positivity to CMA3, AB, and sDF, were observed. It is noteworthy that while motility and viability decreased in almost all subjects, the increase in CMA3 and AB positivity was observed in 68.0% and 79.2% of samples, respectively. A progressive deterioration of sperm motility and viability, less evident for chromatin structure, was observed at longer times of storage (28 and 180 days).Our results indicate that freezing/thawing procedures can alter chromatin structure. A reduction in protamine content and/or a modification in chromatin assembly can be hypothesized. Furthermore, the length of storage in liquid nitrogen appears to progressively affect sperm parameters, although it should be confirmed in larger cohort of subjects.Current sperm cryopreservation protocols need to be improved with new strategies and personalized procedures aimed to minimize the damage. [ABSTRACT FROM AUTHOR]

Germplasm banking is a fundamental tool for the preservation of autochthonous breeds. Semen cryopreservation is effective for this task, but protocols are adapted to commercial species, and post‐thawing sperm quality could be sensitive to environmental cues. We compared the post‐thawing sperm quality in doses from the CBA‐SERIDA bank in northern Spain for the Asturiana de la Montaña (AM) and Asturiana de los Valles (AV) autochthonous cattle breeds. Doses from 23 AM and 16 AV bulls (ejaculates from at least three different seasons) were assessed for motility (computer‐assisted sperm analysis), physiology and chromatin status (flow cytometry) after thawing and after 5 h at 38°C. Data were analysed using linear mixed‐effects and cosinor models for seasonal and breed effects and by correlations with the association of sperm quality with temperature‐humidity index (THI), considering the interval of spermatogenesis plus maturation. The breed affected sperm quality, with higher motility for AV and higher apoptotic ratio, mitochondrial activity, reactive oxygen species, DNA fragmentation and chromatin immaturity for AM. However, seasonality effects were minimal, and THI was not associated with sperm quality. In summary, the season seems to be a minor factor in the post‐thawing quality of the AM and AV autochthonous breeds, well‐adapted to their local environment. [ABSTRACT FROM AUTHOR]

Cryopreservation is a pivotal technique in safeguarding genetic material across diverse species, despite its inherent challenges linked to induced spermatozoa damage, notably apoptosis and lipid peroxidation (LPO). Given the insufficient antioxidant defense of spermatozoa against LPO, there is a rising interest in integrating additional additives into extenders to ameliorate mammalian semen quality. Among these additives, flavonoids have garnered considerable attention due to their potent antioxidative properties. Hence, our study aimed to assess the efficacy of flavone (FL) and 3-hydroxyflavone (3-OH =) supplementation in the cryopreservation medium to protect canine sperm against the damaging impacts of freezing and ensure the preservation of their reproductive potential. Semen was collected from five Beagle stud dogs and then pooled. Then, the sample was divided into 7 groups, each treated with 1) 0 mM, 2) 0.1 mM FL, 3) 0.2 mM FL, 4) 0.4 mM FL, 5) 0.1 mM 3-OH = , 6) 0.2 mM 3-OH = , 7) 0.4 mM 3-OH =. Semen samples were subjected to cryopreservation in French straws and glycerol as a cryoprotectant. In the frozen thawed semen, sperm motility parameters by CASA system and sperm membrane integrity, acrosome status, mitochondrial activity, DNA fragmentation, early apoptosis with capacitation, and LPO were assessed using flow cytometry just after thawing (0 h) and 4 h post thaw. Results reveal significant increase in the proportion of live spermatozoa with undamaged acrosomes in the FL 0.1 and 3-OH = 0.2 groups at 0 h post thaw. At this time point, 3-OH = 0.1 significantly reduced the DNA fragmentation index (DFI) compared to the FL 0.1 and 0.2 groups. However, after the next 4 h, 3-OH = 0.4 exhibited the lowest (P < 0.05) DFI compared to FL 0.2 and 3-OH = 0.1. Additionally, 3-OH = 0.4 showed the highest (P < 0.05) proportion of non apoptotic and non capacitated spermatozoa compared to FL 0.1 0 h post-thaw. Simultaneously, the same group demonstrated significant reduction in apoptotic and capacitated sperm cells, at 0 h and 4 h post-thaw. Moreover, 3-OH = at 0.1 (0 h and 4 h) and 0.2 mM (4 h) significantly enhances the proportion of live sperm without LPO post thaw. Whitin the FL groups, only 0.4 FL significantly increased the percentage of live sperm without LPO. No significant effect of the tested substances was observed on sperm motility, cell membrane integrity, or mitochondrial activity. These findings highlight the promising role of flavone and 3-hydroxyflavone in enhancing sperm resilience during cryopreservation, suggesting their protective function against acrosome damages, capacitation, apoptosis and lipid peroxidation. [Display omitted] • lFlavone (FL) and 3-hydroxyflavone (3-OH =) were used to supplement the extender for canine semen cryopreservation for the first time. • Using FL 0.1 mM and 3-OH = 0.2 mM increased (P < 0.05) the proportion of live spermatozoa with undamaged acrosomes. • Using 3-OH = 0.1 and 0.4 mM significantly reduced the DNA fragmentation index of spermatozoa. • Using 3-OH = 0.4 mM significantly reduced apoptosis and capacitation of sperm cells. • Using FL 0.4 mM and 3-OH = 0.1 and 0.2 mM significantly protected the sperm against lipid peroxidation. [ABSTRACT FROM AUTHOR]

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze–thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze–thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze–thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3–8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at −196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all rs ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial rs ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (rs = 0.366, p = 0.014) and necrosis (rs = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial rs ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (rs = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze–thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood. [ABSTRACT FROM AUTHOR]

Abstract Objective The study aims to assess current international clinician attitudes, practices and barriers towards fertility assessment and preservation in patients undergoing radical inguinal orchidectomy (RIO) for testicular cancer. Materials and methods An international online survey of urologists and urologists in training who perform RIO for testicular cancer was developed by the British Association of Urological Surgeons (BAUS) Sections of Andrology and Oncology and the British Urology Researchers in Surgical Training (BURST). The recruitment process used social media and the emailing lists of national urological societies. Responses were collected between 10/02/2021 and 31/05/2021 and stored using password‐protected Research Electronic Data Capture (REDCap) database software. The primary outcome was the proportion of urologists who routinely offer semen cryopreservation prior to RIO. The study was reported according to the Checklist for Reporting Results of Internet E‐Surveys platform. Results A total of 393 respondents took part in the online survey; of these, the majority were from the United Kingdom (65.9%), with the remaining international respondents (34.1%) from six different continents, which included 45 different countries. Of the respondents, 57.1% reported that they would routinely offer semen cryopreservation to all patients undergoing RIO for testicular cancer. In addition, 36.0% of urologists routinely performed pre‐operative semen analysis, and 22.1% routinely performed pre‐operative testicular serum hormone profile. Of the respondents, 14.4% performed expedited RIO within 48 h; 31.2% of respondents reported that they considered no delay to RIO to allow for semen cryopreservation to be acceptable. Conclusions A significant proportion of international urologists do not offer pre‐operative fertility assessment and preservation in men undergoing RIO for testicular cancer. Surgery is performed in an expedited fashion within 1 week in the majority of patients. Urologists perceive there to be a lack of access and availability to fertility services, and that delay to RIO to allow for fertility preservation is often not acceptable.

OBJECTIVE: To report a novel case of semen cryopreservation after testicular sperm extraction in an adolescent transgender female without cessation of gonadotropin-releasing hormone (GnRH) agonist therapy and feminizing hormone therapy. METHODS: This is a case report of a 16-year-old transgender female using leuprolide acetate for 4 years and estradiol for 3 years requesting semen cryopreservation at the time of gender-affirming orchiectomy. She desired to proceed without cessation of gender affirming hormone therapy. The patients consent was obtained for written publication. RESULTS: The patient underwent testicular sperm extraction followed by orchiectomy. The sample was processed and cryopreserved in a 1:1 Test Yolk Buffer. Multiple early and late spermatids were identified as well as spermatagonium in the TESE specimen. CONCLUSIONS: Advanced spermatogenesis may occur in the presence of a GnRH agonist. Cessation of GnRH agonist therapy may not be essential for semen cryopreservation in adolescent transgender females.

Semen cryopreservation represents a promising technology utilized for preserving high-quality chicken varieties in husbandry practices. However, the efficacy of this methodology is significantly impeded by the diminished quality of sperm. Metabolites, as the end products of metabolic reactions, serve as indicators of biological processes and offer insights into physiological conditions. In this study, we investigaged the sperm quality and alteration in metabolic profiles during the cryopreservation of Longyou Partridge Chicken semen. Following artificial semen collection, four groups of semen samples were established based on four points of the cryopreservation process (Ⅰ, fresh semen; Ⅱ, semen added extender and chilled at 4 °C for 30 min; Ⅲ, semen added cryoprotectants; Ⅳ, semen gradient freezed and stored in liquid nitrogen). Semen cryopreservation has a negative effect on the percentage of sperm in a straight-line trajectory (LIN), has no significant effect on total motile sperms (TM) or the proportion of sperm with typical morphology (NM). Metabolites were identified using LC-MS technique and analyses including Principal Component Analysis (PCA), Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), Univariate statistical analysis, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were employed to identify metabolites. A total of 2471 metabolites had been identified, with the majority of the list being made up of amino acids and their metabolites as well as benzene and substituted derivatives. Group II exhibits 882 metabolites with significantly elevated abundance relative to Group I, alongside 37 metabolites displaying decreased abundance. In Group III, 836 metabolites demonstrate notably augmented abundance compared to Group II, while 87 metabolites exhibit reduced abundance. Furthermore, Group IV showcases 513 metabolites with markedly heightened abundance in comparison to Group III, and 396 metabolites with decreased abundance. Specific metabolites such as 5-Hydroxylysine, Phosphocholine, and alpha- d -glucose-6-phosphate exhibited a progressive decline during the cryopreservation process, correlating with either dilution and chilling, cryoprotectant addition, or freezing. In conclusion, our investigation systematically examined the changes of seminal metabolome and sperm quality throughout the cryopreservation process of rooster semen. • The alterations in metabolites during cryopreservation of rooster semen were investigated through four pivotal stages. • It demonstrates correlations between the semen metabolome and sperm quality. • Certain metabolites were newly found to decline progressively during semen cryopreservation. [ABSTRACT FROM AUTHOR]

Simple Summary: Liquid nitrogen (LN) is usually used for semen cryopreservation. The most significant point is that it has no time or space restrictions, maximizing the use of high-quality males for breeding. Dry ice is also one of the earliest media used in animal semen cryopreservation. The efficiency of dry ice cryopreservation is lower than that of LN. However, compared with LN, dry ice has an advantage in transportation, as it permits express delivery and can be carried on trains and high-speed railways. Therefore, using dry ice as a cold source for preserving and transporting ultralow-temperature-preserved semen liquid has received extensive attention from scholars. It has been demonstrated in frozen semen from mice and cattle that dry ice can be used as a reliable cold source for transportation. For this reason, we simulated a dry ice transportation environment by placing ultralow-temperature-frozen semen in a foam box with sufficient dry ice to maintain it for one week. We found that one week of storage in dry ice did not cause significant damage to porcine spermatozoa. The effects on the semen were essentially the same as those of LN storage when the dry-ice-preserved spermatozoa were transferred to LN again after one week. To address the safety problems posed by the transportation of boar semen using LN, this study was conducted on the short-term storage of frozen boar semen in dry ice (−79 °C). Boar semen frozen in LN was transferred to dry ice, kept for 1 day, 3 days, 5 days, 7 days, or 8 days, and then moved back to LN. The quality of frozen semen stored in LN or dry ice was determined to evaluate the feasibility of short-distance transportation with dry ice. The results showed that 60 °C for 8 s was the best condition for thawing frozen semen stored in dry ice. No significant differences in spermatozoa motility, plasma membrane integrity, or acrosome integrity were observed in semen after short-term storage in dry ice compared to LN (p > 0.05). There were no significant changes in antioxidant properties between storage groups either (p > 0.05). In conclusion, dry ice could be used as a cold source for the short-term transportation of frozen boar semen for at least 7 days, without affecting sperm motility, morphological integrity, or antioxidant indices. [ABSTRACT FROM AUTHOR]

Simple Summary: The conservation of local chicken breeds is globally considered because of their interesting characteristics, such as their tolerance to heat stress, resistance to diseases and capacity to survive in severe environments. In recent years, conservation programs of native chicken breeds have been developed in Italy, and this work has focused on Bionda Piemontese, Bianca di Saluzzo and Pepoi, the first two of which are catalogued as endangered and the third as a 'critically maintained' breed. Semen cryopreservation is the only method that is currently feasible for the ex situ management of genetic diversity in birds, and poultry sperm shows a greater susceptibility during the freezing/thawing process due to its unique features. Therefore, the choice of the most suitable rooster sperm donor is fundamental in the establishment of a semen cryobank. The present study aims to analyze the efficiency of bird and semen management required for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds created with the "TuBAvI" project. Our results suggest that male donors need to be selected based on the quality of fresh ejaculates, regardless of sperm outcome after freezing/thawing, in order to identify individuals who can ensure the survival of a breed with regard to increased fertilizing capacity. Native breed conservation is an important component of poultry biodiversity. The aim of this work is to describe different steps that lead to donor selection for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. The variability within and between breeds was evaluated, and the stored semen reproductive capacity was in vivo tested using artificial insemination. Semen from Bionda Piemontese, Bianca di Saluzzo and Pepoi roosters was collected and processed. Concentration, volume, sperm membrane integrity, total motile sperm, progressive motile sperm and kinetic parameters were analyzed; sperm parameters accounting for bird variability were used to select male donors. Fresh semen quality parameters measured in donor ejaculates showed significant differences between breeds; no differences were found after cryopreservation. Variability in the fertilizing ability of cryopreserved semen was found within a breed (5–16%) and between birds within a breed (BP = 3–7%; BS = 7–31%; PP = 6–22%); only sperm quality parameters measured in fresh ejaculates, not frozen/thawed, may be associated with in vivo fertility results. In conclusion, sperm concentration and progressive motility were successfully used as selection parameters to identify chicken male donors with improved sperm quality for sperm cryobanking. However, new reliable sperm markers to predict cryopreserved semen's fertilizing ability are required. [ABSTRACT FROM AUTHOR]

Oncofertility, an interdisciplinary field, aims at improving the quality of life of cancer patients prone to iatrogenic infertility by offering fertility preservation options for a potentially fertile future. Semen cryopreservation (SC) is a well-established and effective way of preserving fertility in adolescent and young adult cancer-diagnosed males as they face potential fertility loss due to cancer and its treatments. The pathophysiology of certain cancers, such as testicular cancer or Hodgkin's lymphoma, has been shown to have an adverse effect on semen quality. Ablative therapies like chemotherapy and radiotherapy can result in compromised sperm parameters depending on the dose and the type of the drug or radiation. Hence, it is recommended to encourage cancer-diagnosed males to store sperm prior to gonadotoxic therapy to restore potential fertility in the future. Despite the feasibility of SC, this option remains underutilised due to several limitations. [ABSTRACT FROM AUTHOR]

Reduced glutathione (GSH) is a natural redox force immediately consumed by cells to combat toxins. Today's world is shifting towards clean farming practices whence ring‐necked pheasants dependent on croplands are facing threats and sperm‐assisted reproductive techniques become indispensable in preserving this valuable species for game and meat purposes. This study aims at evaluating the cryosurvivability (motility, viability, plasma membrane and acrosomal integrity, DNA fragmentation) and antioxidant capacity (Free radical scavenging activity, LPO detection, Ferric reducing antioxidant power, mitochondrial status) of ring‐necked pheasant semen with GSH supplementation in the extender. Semen collected from ten males was pooled, divided and diluted with Beltsville poultry semen extender (1:5) containing 0.0, 0.2, 0.4, 0.6 and 0.8 mM GSH. Samples were cryopreserved, and sperm quality was evaluated post‐dilution, cooling, equilibration, and freeze–thaw. Free radical scavenging activity, total antioxidant potential, mitochondrial action and lipid peroxidation were assessed at post‐thawing. Sperm motility, plasma membrane and acrosomal integrity, viability and DNA integrity were recorded highest (p <.05) at 0.4 mM GSH compared to other concentrations and control at all the stages of cryopreservation. Similarly, sperm mitochondrial action, antioxidant potential and free radical scavenging capacity were recorded higher (p <.05) with 0.4 mM GSH at post‐thaw compared to other concentrations of GSH and control. Lipid peroxidation was recorded as highest (p <.05) in the control compared to GSH‐supplemented groups at post‐thaw. Hence, it is concluded that GSH at 0.4 mM preserves the quality and ROS scavenging capacity of cryopreserved ring‐necked pheasant sperm as an antioxidant. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate alpaca pregnancy outcomes and birth rates of females inseminated with frozen semen using two commercial extenders. A total of 18 ejaculates from 8 adult alpaca males were obtained with artificial vagina, and macroscopic and microscopic semen characteristics were assessed. Afterwards, samples were divided into two aliquots, diluted with Biladyl® B or AndroMed®, and cooled for 2 h at 5°C. At that moment, sperm motility was evaluated, and samples were frozen through a gradual descent of temperature using a liquid nitrogen tank. To analyse frozen sperm quality, samples were thawed at 38°C for 30 s. Even though a significant decrease in sperm motility and viability was detected when thawed (p <.05), no superiority was found between the two commercial extenders (Biladyl® B vs. AndroMed®). A total of 36 alpaca females were artificially inseminated (AI) between 30 and 34 h post‐injection of a GnRH analogue, administered when a growing dominant follicle was detected through transrectal palpation and ultrasonography. Obtained pregnancy rates were similar between Biladyl® B (33.3%, 6/18) and AndroMed® (22.2%, 4/18). No significant differences were detected in birth rates between the two tested extenders, obtaining 4 and 3 births for Biladyl® and AndroMed®, respectively. In conclusion, alpaca pregnancies and alive offspring can be obtained through AI with frozen semen at similar efficiency rates using commercial diluents, Biladyl® B or AndroMed®. [ABSTRACT FROM AUTHOR]

Poultry semen cryopreservation technology is one of the most direct methods to protect poultry germplasm, and in this study, the back and abdomen massage method is used to collect the semen of 20 Huoyan male geese, which is on the basis of EK diluent adding different combinations of dimethyl sulfoxide (DMSO) and trehalose, in total 7 different combinations treatments were achieved: D1T0 (5%DMSO), D1T1 (5% DMSO + 50 mM trehalose), D1T2 (5%DMSO + 100 mM trehalose), D2T0 (10% DMSO), D2T1 (10% DMSO + 50 mM trehalose), D2T2 (10% DMSO + 100 mol trehalose) and no addition group D0T0 (0% DMSO + 0 mM trehalose). Diluted semen was frozen in granules in liquid nitrogen vapour. After thawing in 40°C water bath, determinate sperm livability, acrosome integrity, plasma membrane integrity, catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, malondialdehyde (MDA) content and reactive oxygen species (ROS) level, and the results showed that 10% DMSO and 0.05 mol/L trehalose could significantly improve the sperm motility, acrosome integrity rate and plasma membrane integrity rate after thawing, and significantly increase the levels of antioxidant enzymes CAT, SOD and GSH-Px in seminal plasma (P

To preserve male fertility after diagnosis of any kind of cancer, a prompt assessment of the semen quality and an appropriate semen cryopreservation must be performed before radio-chemotherapy starts. The present work aims to evaluate the semen parameters at diagnosis of different cancer patients before cryopreservation and after thawing. Testicular tumors and lymphomas are among the most common cancers in younger patients, and while chemotherapy significantly increases patients' survival, it can epigenetically alter the semen fluid, resulting in temporary or permanent infertility. We analyzed data from the database of the Gamete Cryopreservation Center (Annunziata Hospital, CS; Italy) in the period of 2011–2020 from a cohort of 254 cancer patients aged 18–56 years. The evaluation was performed in a blind manner and anonymously recovered; the main parameters referring to semen quality were assessed in accordance with the WHO guidelines and decision limits (6th edition; 2021). The cancer types were as follows: testis cancers (TC; n = 135; 53.1%), hematological cancers (HC; n = 76; 29.9%), and other types of cancer (OC; n = 43; 17%). Comparing TC vs. HC (P1) and vs. OC (P2), TC had the worst semen quality: sperm number/mL (P1 = 0.0014; P2 = 0.004), total motility (P1 = 0.02; P2 = 0.07), progressive motility (P1 = 0.04; P2 = 0.05), viability (P1 = 0.01; P2 = 0.02), and percentage of atypical morphology (P1 = 0.05; P2 = 0.03). After semen thawing, viability and progressive motility recovery lowered, accounting for 46.82% and 16.75%, respectively, in the whole cohort; similarly, in the subgroups ascribed to TC, they showed the lowest recovery. Strong correlation existed between pre- and post-cryopreservation viability and progressive motility in the whole cohort (p < 0.001) and in the TC subgroup (p < 0.05). All cancer subgroups, to significantly different extents, had semen findings below the WHO reference values, suggesting diverse sperm susceptibilities to different cancers and cryodamage. Cancer and associated treatments epigenetically affect patients' semen quality, meaning cryopreservation should be considered a useful personalized prerogative for any kind of cancer in a timely manner. [ABSTRACT FROM AUTHOR]

Sperm preservation is a well‐established technique in reproductive biotechnology that is widely used to maintain the genetic quality of male individuals. However, there are several factors during the preservation process that can affect the vitality, functionality, and quality of sperm, thereby reducing their fertility potential after thawing. One of these factors is the synthesis of high levels of oxidative stress (OS) during semen preservation, which can have detrimental effects on sperm health and functionality. To counter the negative impact of OS on sperm, researchers have explored the supplementation of several exogenous antioxidants in the extenders used to preserve ram sperm. This approach has shown promising results in improving sperm health, functionality, and fertility potential in ram. Additionally, the preservation process can induce modifications in the ram sperm proteome. By employing targeted proteomics techniques, researchers have been able to identify and modify specific proteins in cryopreserved ram sperm, potentially offering further improvements in the quality of the cryopreserved ram sperm. In summary, this review provides a comprehensive overview of the antioxidants and targeted proteomics modifications that have been investigated for enhancing ram sperm preservation. These advancements aim to mitigate the negative effects of OS and optimize the techniques used in preserving ram sperm. [ABSTRACT FROM AUTHOR]

As the number of cancer survivors increases, so does the demand for preserving male fertility after radiation. It is important for healthcare providers to understand the pathophysiology of radiation-induced testicular injury, the techniques of fertility preservation both before and during radiation, and their role in counseling patients on the risks to their fertility and the means of mitigating these risks. Impaired spermatogenesis is a known testicular toxicity of radiation in both the acute and the late settings, as rapidly dividing spermatogonial germ cells are exquisitely sensitive to irradiation. The threshold for spermatogonial injury and subsequent impairment in spermatogenesis is ~ 0.1 Gy and the severity of gonadal injury is highly dose-dependent. Total doses < 4 Gy may allow for recovery of spermatogenesis and fertility potential, but with larger doses, recovery may be protracted or impossible. All patients undergoing gonadotoxic radiation therapy should be counseled on the possibility of future infertility, offered the opportunity for semen cryopreservation, and offered referral to a fertility specialist. In addition to this, every effort should be made to shield the testes (if not expected to contain tumor) during therapy. [ABSTRACT FROM AUTHOR]

The current study aimed to evaluate the impact of different antioxidants on the sperm quality-related parameters of frozen-thawed Brown Swiss bulls. Semen samples were collected from each bull once per week using an artificial vagina. The semen samples were pooled and divided into aliquots. One of them was diluted with a tris-based extender without any antioxidants and served as a control group. Other aliquots were diluted with a tris-based extender supplemented with 0.75% green tea extract, 0.25% strawberry extract, 1.5 mM of vitamin E, 2 mM of vitamin C, and 0.5 mM of caffeine at 37°C. The semen was evaluated for motility, viability, abnormality, plasma membrane integrity, and acrosome integrity immediately following the addition of diluent for the control group and again after one week of storage in liquid nitrogen at -196°C and re- thawing for all groups. The results revealed that green tea extract had the most beneficial impact on improving sperm parameters (motility, viability, abnormality, plasma membrane integrity, and acrosome integrity) followed by strawberry extract and vitamin E then vitamin C and caffeine as compared to the post-thawing control group. So, according to the results obtained, it was concluded that addition of green tea extract in the semen extender can enhance the quality of frozen-thawed bull sperm. However, given the price and availability of strawberry, we recommend using strawberry extract to boost seminal quality. [ABSTRACT FROM AUTHOR]

To address the safety problems posed by the transportation of boar semen using LN, this study was conducted on the short-term storage of frozen boar semen in dry ice (−79 °C). Boar semen frozen in LN was transferred to dry ice, kept for 1 day, 3 days, 5 days, 7 days, or 8 days, and then moved back to LN. The quality of frozen semen stored in LN or dry ice was determined to evaluate the feasibility of short-distance transportation with dry ice. The results showed that 60 °C for 8 s was the best condition for thawing frozen semen stored in dry ice. No significant differences in spermatozoa motility, plasma membrane integrity, or acrosome integrity were observed in semen after short-term storage in dry ice compared to LN (p > 0.05). There were no significant changes in antioxidant properties between storage groups either (p > 0.05). In conclusion, dry ice could be used as a cold source for the short-term transportation of frozen boar semen for at least 7 days, without affecting sperm motility, morphological integrity, or antioxidant indices.

Background: Sperm cryopreservation is recommended to preserve male fertility for cancer patients or other medical conditions at risk of sperm decline. Whether motility and viability recovery rates vary depending on the medical conditions requiring cryopreservation is poorly known. We report here on the 24-year experience of our semen bank. Methods: Motility and viability recovery rates were evaluated in 1973 collections from patients with various medical conditions and 67 collections from donors, and the results were related to basal semen quality. Results: Motility and viability recovery were highly related to basal semen quality and varied between cancer and non-cancer conditions, independently of the duration of cryopreservation and patient age. In samples with a sperm number below 2 × 106/mL, recovery rates approximated to zero. The highest recovery rates were found in donor collections. Cut-off values for the recovery of at least 1% motile spermatozoa were established based on initial semen quality. Conclusions: Our results indicate that the occurrence of any pathological or medical condition resulted in lower recovery rates with respect to donors, indicating that intrinsic sperm characteristics drive susceptibility to cryodamage. Established cut-off values for motility recovery can be useful for patient counseling as well as for ART laboratories to decide the type of procedure. [ABSTRACT FROM AUTHOR]

The objectives of this study were to assess the cooling and freezing of donkey epididymal semen harvested immediately after castration (Experiment 1, n = 4) or after the shipment (24 or 48 h) of epididymides attached to testicles (Experiment 2, n = 14) or dissected apart (Experiment 3, n = 36). In each experiment, semen was frozen immediately (Non-Centrif) in an egg yolk-based semen extender (EY) or after processing through cushion-centrifugation (Centrif) while extended in a skim milk-based extender (SC). In all three experiments, cooled, pre-freeze, and post-thaw epididymal semen was assessed for total motility (TM), progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP). Data were analyzed with R using mixed models and Tukey's test as posthoc. Results showed that the cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity; furthermore, in Experiment 3, the post-thaw evaluation of both Centrif and Non-Centrif achieved similar TM and PM. Collectively, the post-thaw results revealed low motility parameters across groups; while, the PMI and HMMP did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation with either centrifugation or extenders. In summary, freshly harvested and cooled-shipped and cooled semen had satisfactory semen parameters. Future studies need to address donkey epididymal semen fertility in mares and jennies.

Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.

Résumé Contexte La cryoconservation des spermatozoïdes est une méthode efficace de préservation de la fertilité pour la congélation des spermatozoïdes liée à des causes médicales et aux demandes sociétales. Au total, 662 hommes (entre 15 et 65 ans; moyenne: 33,5 ± 8,8 ans) ont été inclus dans cette étude pour évaluer les caractéristiques de la population, la qualité du sperme et l’utilisation de la préservation autologue de spermatozoïdes réalisée par des patients à Beijing. Parmi ceux-ci, 351 étaient des patients atteints de cancer (53%; 31,1 ± 7,3 ans) et 311 des patients non atteints de cancer (47%; 36,1 ± 9,5 ans). Résultats Nous avons constaté que le nombre de cas de conservation a augmenté régulièrement de 2015 à 2019; 89,7% d’entre eux avaient un baccalauréat ou plus; 54,8%, 41,5% et 3,6% étaient respectivement célibataires, mariés, ou divorcés. Les cas de cancer et ceux d’oligozoospermie représentaient 71,3% de tous les patients; par conséquent, la plupart des patients avaient besoin d’une préservation de la fertilité pour raison de maladie. Le groupe des hommes atteints de cancer avait significativement une plus faible concentration de spermatozoïdes, un plus faible taux de spermatozoïdes progressifs après le test de congelation-décongelation, un plus faible nombre total de spermatozoïdes de motilité progressive après le test de congelation-décongelation, et un plus faible taux de récupération de spermatozoïdes mobiles progressifs (TRMP) que le groupe de patients non atteints de cancer (tous p

In this study, we explore the effects of Poria cocos mushroom polysaccharides (PCPs) on the quality and DNA methylation of the cryopreserved spermatozoa of Shanghai white pigs. A total of 24 ejaculates (three ejaculate samples per boar) from eight Shanghai white pigs were manually collected. The pooled semen was diluted with a based extender supplemented with different concentrations of PCPs (0, 300, 600, 900, 1200, and 1500 μg/mL). Once thawed, the quality of the spermatozoa and their antioxidant function were assessed. In the meantime, the effect of spermatozoa DNA methylation was also analyzed. The results show that compared with the control group, 600 μg/mL of PCPs significantly improves the spermatozoa viability (p < 0.05). The motility and plasma membrane integrity of the frozen–thawed spermatozoa are significantly higher after treatment with 600, 900, and 1200 μg/mL of PCPs compared with the control group (p < 0.05). In comparison with the control group, the percentages of acrosome integrity and mitochondrial activity are significantly enhanced after the application of 600 and 900 μg/mL PCPs (p < 0.05). The reactive oxygen species (ROS), the malondialdehyde (MDA) levels, and the glutathione peroxidase (GSH-Px) activity, in comparison with the control group, are significantly decreased in all groups with PCPs (all p < 0.05). The enzymatic activity of superoxide dismutase (SOD) in spermatozoa is significantly higher in the treatment with 600 μg/mL of PCPs than in the other groups (p < 0.05). As compared with the control group, a significant increase in the catalase (CAT) level is found in the groups with PCPs at 300, 600, 900, and 1200 μg/mL (all p < 0.05). In comparison with the control group, the 5-methylcytosine (5-mC) levels are significantly decreased in all groups with PCPs (all p < 0.05). As a result of these findings, a certain amount of PCPs (600–900 μg/mL) added to the cryodiluent can significantly improve the quality of Shanghai white pig spermatozoa and can also reduce the methylation of spermatozoa DNA caused by cryopreservation. This treatment strategy may establish a foundation for the cryopreservation of semen from pigs. [ABSTRACT FROM AUTHOR]

Simple Summary: Germplasm banks provide an essential service by safeguarding the genetics of endangered or valuable species or individuals. The SERIDA (Asturias, Spain) keeps germplasm from several valuable local breeds. This study aimed to characterize the viability of the cryopreserved semen samples from the "Asturiana de la Montaña," a valuable autochthonous breed adapted to the mountainous Atlantic environment. We assessed the sperm quality of the semen doses from 40 bulls after thawing. We combined this data with fresh semen quality and fertility data from females inseminated with semen doses from these bulls. Results showed that "Asturiana de la Montaña" bulls produce semen of comparable quality to commercial breeds, fresh and frozen-thawed, confirming the viability and usefulness of the SERIDA germplasm for preserving and spreading this breed's genetics. The fertility of the semen doses was also acceptable. However, we did not find a relationship between the semen quality results and the field fertility, except for the linearity of the spermatozoa (proportion swimming in more linear paths). Whereas the sperm parameters of this endangered breed are acceptable, future research should focus on optimizing the existing protocols for further improving post-thawing semen quality. Semen cryobanks are critical for preserving autochthonous and rare breeds. Since sperm cryopreservation has been optimized for commercial breeds, non-commercial ones (often endangered) must be characterized to ensure the germplasm's viability. This study reports an investigation of the "Asturiana de la Montaña" breed (AM), a valuable Spanish autochthonous cattle breed adapted to the mountainous Atlantic environment. The survey included cryopreserved semen doses from 40 bulls stored at the Principado de Asturias Germplasm Bank. Data were obtained from the routine fresh semen analysis, CASA (motility), and flow cytometry analyses of fresh and post-thawing semen, and the 56-day non-return-rate (NRR) in heifers and cows (all results as 1st and 3rd quartiles). Fresh samples (artificial vagina) were within the normal range for cattle (4–6 mL, 5–10 × 109/mL; mass motility 5). Post-thawing results showed motility below typical for commercial breeds (total motility 26–43%, progressive 14–28%), with higher values for viability (47–62%). Insemination results showed a good performance for this breed (NRR: 47–56%; higher for heifers). Sperm volume increased with age, with little or no effects on sperm quality. Few associations were found between post-thawing quality or freezability and NRR, LIN being the variable more strongly associated (positively). The AM semen bank shows a good prospect for preserving and disseminating the genetics of this breed. This survey indicates that dedicated research is needed to adapt freezing protocols to this breed, optimizing post-thawing results. [ABSTRACT FROM AUTHOR]

Giant grouper (Epinephelus lanceolatus) is not only favored in the international live reef-fish trade but also a promising species for mariculture in coastal communities throughout Asia. Sperm cryopreservation contributes to the extensive utilization of artificial insemination in grouper aquaculture industry. Inevitable damage during the process of freezing can negatively affect post-thaw sperm quality and fertility, thus hindering its routine application in practice. Melatonin is an antioxidant that can protect sperm from the adverse effects of cryopreservation in a variety of species. This study was aimed to assess the effect of melatonin addition on post-thaw sperm function of giant grouper. Semen samples were cryopreserved with freezing medium containing different final concentrations of melatonin (1, 2 and 4 μM) and the control group (without melatonin). After thawing, multiple sperm functional parameters were evaluated. Generally, a positive effect on cryopreservation was observed for all doses of melatonin. Melatonin treatment at 1 and 2 μM seemed to be efficient in preventing sperm cryodamage, which yielded significantly higher total motility, progressive motility, mitochondrial membrane potential (P

Native breed conservation is an important component of poultry biodiversity. The aim of this work is to describe different steps that lead to donor selection for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. The variability within and between breeds was evaluated, and the stored semen reproductive capacity was in vivo tested using artificial insemination. Semen from Bionda Piemontese, Bianca di Saluzzo and Pepoi roosters was collected and processed. Concentration, volume, sperm membrane integrity, total motile sperm, progressive motile sperm and kinetic parameters were analyzed; sperm parameters accounting for bird variability were used to select male donors. Fresh semen quality parameters measured in donor ejaculates showed significant differences between breeds; no differences were found after cryopreservation. Variability in the fertilizing ability of cryopreserved semen was found within a breed (5–16%) and between birds within a breed (BP = 3–7%; BS = 7–31%; PP = 6–22%); only sperm quality parameters measured in fresh ejaculates, not frozen/thawed, may be associated with in vivo fertility results. In conclusion, sperm concentration and progressive motility were successfully used as selection parameters to identify chicken male donors with improved sperm quality for sperm cryobanking. However, new reliable sperm markers to predict cryopreserved semen’s fertilizing ability are required.

Background: Semen cryopreservation could be enhanced by integration of biocompatible nanomaterials. Conditions in which nanoparticles are synthesized should be taken in consideration to avoid divergences in research results concerning beneficial or toxic effects. The objective of this study was to examine the potential of zinc oxide nanoparticles (ZnONPs) synthesized by simple co-precipitation method for providing protection to buffalo sperm against cryo-damage. Methods: ZnONPs were characterized by means of X-ray diffraction, high resolution transmission electron microscope (HR-TEM), zetasizer and Raman spectroscopy. Semen was collected from six healthy buffalo bulls using artificial vagina and pooled semen samples were diluted with tris-egg yolk-fructose extender only (control) or extender supplemented with 10, 25, 50, 100 and 200 µg/ml of ZnONPs. Results: Sperm motility, membrane integrity and acrosome defects were measured. DNA damage of spermatozoa was evaluated using comet assay. To determine antioxidative role of ZnONPs, malondialdehyde (MDA) level as a biomarker of lipid peroxidation was measured in seminal plasma. Sperm motility, viability index as well as membrane integrity increased with ZnONPs concentrations up to 100 μg/ml. Likewise, no adverse effect was reported on sperm DNA integrity with all concentrations. MDA decreased up to concentration of 100 μg/ml ZnONPs. Notably, adding 25, and 50 μg/ml ZnONPs to extender produced best significant positive effects on semen parameters compared to ZnONPs free group. Conclusion: The results confirm that low concentrations of ZnONPs fabricated by co-precipitation method using water as exchange media could effectively preserve the quality of buffalo bull frozen-thawed sperm by reduction of cryoinjury.

The mitochondria-targeted antioxidant MitoQ has been regarded as an effective antioxidant agent against cryo-induced oxidative cellular damage. This study aimed to evaluate the use of different doses of MitoQ combined with trehalose to minimize mitochondrial impairment and oxidative stress during sperm cryopreservation of Markhoz goat. For this, semen samples (n = 50) were collected by electroejaculation every 5 days from 5 bucks in 10 replicates. On each collection day, 5 ejaculates (one ejaculate for each buck) were pooled and then diluted in eight different Tris-based extenders as follows: no additives (control), 20, 200, 2000 nM of MitoQ (MT20, MT200, MT 2000, respectively), 150 mM of trehalose (Tr), MT20+Tr, MT200+Tr, MT2000+Tr. The semen samples were frozen using a standard protocol, and sperm function and oxidative stress were evaluated after thawing. The semen extender supplemented with MT200+Tr had higher (P < 0.05) total and progressive motility, acrosome and membrane integrity, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and lower (P < 0.05) DNA fragmentation, malondialdehyde and intracellular hydrogen peroxide levels than the all other groups except MT200; meanwhile, MT200 was also improved (P < 0.05) in these parameters than in the control group. Furthermore, MT200 and MT200+Tr showed higher (P < 0.05) percentages of live cryopreserved sperm with high mitochondrial activity than other groups. However, abnormality percentage and catalase activity of frozen-thawed sperm were not affected by treatments (P > 0.05). To conclude, we have found that supplementation of 200 nM MitoQ alone or in combination with 150 mM trehalose to semen extender improved the quality of cryopreserved sperm in goats, which is associated with enhanced antioxidant enzymatic defense and mitochondrial activity and reduced DNA fragmentation. [ABSTRACT FROM AUTHOR]

Simple Summary: This study mainly aimed to evaluate the seasonal and breed effects on spermatozoa (SPZ) traits of ten Portuguese sheep breeds, cryopreserved and stored in the Portuguese National Germplasm Bank. The mean volume and SPZ concentration of the ejaculate was 0.77 mL and 5039 ± 51 × 106/mL, respectively, and were affected by breed but not by season. The viability (alive SPZ), individual motility, and abnormal morphology of fresh semen were 63.2 ± 0.2%, 61.41 ± 0.2%, and 10.1 ± 0.1%, respectively. A significant decrease of 28.5% of the viability and 35.5% of individual motility, as well as an increase of 54.5% in the SPZ abnormal morphology was observed in thawed semen. An effect of breed, but not of season, was observed on these SPZ traits. Nonetheless, breed × season interactions influenced individual motility and abnormal morphology, except for tail defects. Overall, the effect of season on ejaculate and SPZ traits was not significant and local rams are able to be SPZ donors for cryopreservation during the whole year in our latitudes. The semen quality is one of the determinant factors of ram semen cryopreservation. The present retrospective study aimed to characterize the seasonal ram pattern during the year for ten Portuguese local sheep breeds, hypothesizing that the breed and season had low effects on the main spermatozoa traits. A total of 1471 ejaculates were used and evaluated (fresh semen) from 85 rams between 2004 and 2020 and re-evaluated after thawing (thawed semen). The effect of breed, season, and sperm cryopreservation on nine semen traits were evaluated. The volume per ejaculate, spermatozoa (SPZ) concentration, and total number of SPZ per ejaculate, were affected by breed (p < 0.001) but not by season (p > 0.05). As expected, the semen processing was the most significant (p < 0.001) factor of variation on seminal parameters. Moreover, breed and interactions between breed × semen processing, modulated the response of alive SPZ, abnormal morphology, head, and intermediate piece defects. In fresh semen, season only affected the intermediate piece defects due to the highest percentage observed between February and April period in some breeds. Overall, and despite the mentioned particularities, there were similarities among the ten local breeds. We also concluded that the seasonal effect on ejaculate and SPZ traits is not significant in our region. These local ram breeds have low seasonality and can be employed in natural mating as well as semen donors for cryopreservation and assisted reproductive biotechnologies during the whole year at our latitude. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of melatonin (MLT), reduced glutathione (GSH) and vitamin E (Vit. E) or their combinations on semen cryopreservation of Mediterranean buffalo. The quality parameters such as viability, abnormality rate, motility, structural integrity and the antioxidant capacity of frozen-thawed sperm were evaluated. The efficiency of frozen-thawed sperms in performing their functions was further analyzed by in vitro fertilization (IVF). In those separately supplemented groups, 0.2 mg/mL MLT, 0.2 mM GSH and 0.4 mg/mL Vit. E had the best effect on antioxidant capacity, kinetics and morphology, respectively. In addition, the cleavage, blastocyst and hatching blastocyst rates of IVF embryos were higher in 0.2 mg/mL MLT, 0.2 mM GSH, 0.2 and 0.4 mg/mL Vit. E groups than the blank control. Among the three combination groups, the kinetics and structure integrity of frozen-thawed sperms, cleavage, blastocyst and hatching blastocyst rates of IVF embryos were higher in 0.4 mg/mL Vit. E plus 0.2 mg/mL MLT group than the blank control group, revealed that this combination had comprehensive protection on frozen-thawed sperm of Mediterranean buffalo. These results support to develop special semen freezing extender containing an optimal choice of MLT, GSH and Vit. E, and to enhance the efficiency of frozen-thawed sperm of Mediterranean buffalo for IVF. • MLT had better antioxidant activity than GSH and Vit. E in sperm cryopreservation. • The 0.4 mg/mL Vit. E + 0.2 mg/mL MLT showed highest hatching rate of IVF embryos. • The combination of 0.4 mg/mL Vit. E + 0.2 mg/mL MLT had comprehensive protection. [ABSTRACT FROM AUTHOR]

Simple Summary: The current study aimed to compare the effects of the replacement of egg yolk in an extender with gum arabic along with supplementation with cysteine or ascorbic acid on semen quality and freezability in Noemi rams in vitro. Semen from six rams were collected with an artificial vagina two times per week. Spermatozoa motility and concentration were estimated with the computer-assisted semen analysis system. The semen samples were frozen using a Tris extender containing either egg yolk or gum arabic and supplemented with different concentrations of cysteine or ascorbic acid as antioxidants. The semen from each ejaculate of each ram were resuspended with a specific extender with glycerol for cryopreservation. The addition of antioxidants to both extenders has advantageous effects; however, supplementation of gum arabic extender with antioxidants cysteine (0.5 or 1 mM) or ascorbic acid (0.5 mM) has beneficial effects on semen quality after cryopreservation. Semen cryopreservation is very important in animal agriculture to maximize the number of daughters of genetically superior males and to distribute the cryopreserved semen of good males all over the world. However, the freezing process generates some damage to sperm that reduce their fertilizing ability after thawing. Moreover, egg yolk, which is the most common animal-origin cryoprotectant used in semen dilution, is considered a source of biosecurity risk. In the current study, we aimed to compare the replacement of egg yolk in the extender by gum arabic (5%) along with supplementation with antioxidant cysteine or ascorbic acid on semen quality and freezability in Noemi rams in vitro. Semen from six rams were collected with an artificial vagina two times per week. Semen evaluation parameters such as color, volume, pH, general motility, percentage motility, concentration and cell viability ratio were assessed. Spermatozoa motility and concentration were estimated with the computer-assisted semen analysis system. The semen samples were frozen using a Tris extender containing either 15% egg yolk or 5% gum arabic. For antioxidant-supplemented extenders, cysteine or ascorbic acid was dissolved at concentrations of 0.10, 0.50 or 1.0 mM in egg yolk or gum arabic extender. The semen from each ejaculate of each ram were resuspended with a specific extender with glycerol (5%); the final volume after dilution was 1 mL semen to 4 mL extender. The samples were then cooled to 4 °C for 120 min, loaded into 0.5 mL straws and frozen in liquid nitrogen for 7 days. Supplementation of gum arabic or egg yolk extenders for ram semen with antioxidants such as cysteine or ascorbic acid has beneficial effects on semen quality after cold storage or cryopreservation. However, supplementation of a 5% gum arabic extender with cysteine at 0.5 or 1 mM concentration or ascorbic acid at 0.5 mM concentration improved the quality of spermatozoa postcryopreservation. It could be concluded that gum arabic is a good alternative for egg yolk in Noemi ram semen extenders. Antioxidants are necessary to support the addition of gum arabic to the extender to help the ram spermatozoa to survive freezing–thawing and oxidative stresses. [ABSTRACT FROM AUTHOR]

Objective To investigate the protective effects of nicotinamide adenine dinucleotide (NAD) and its precursor nicotinamide adenine mononucleotide (NAM) on human frozen sperm motility, DNA and acrosin activity. Methods Total of 50 male semen samples were collected from reproductive medicine center of our hospital in the study, the spermmotility, DNA fragmentation index (DFI) and acrosin activity were detected. All the semen samples were classified into prefreezing intervention group and post-resuscitation intervention group according to the different added time of NAD / NAM. The samples of the pre-freezing intervention group were further divided into three subgroups: they were treated with PBS, NAD and NAM, respectively and incubated at 37 °C for 30 min. The sperm motility (PR level), DFI and acrosin activity were detected. After frozen with cryoprotectant for more than 24 h, the three indexes in samples were detected again. The samples in the post resuscitation intervention group were directly added with sperm cryoprotectant, frozen for more than 24 hours, and then resuscitated. The recovered samples were divided into three parts: and they were treated with PBS, NAD and NAM and incubated at 37 七 for 30 min, and then the sperm motility, DFI and acrosin activity were detected. Additionally, 38 male semen samples were collected, each case was divided into two parts. One was resuscitated after 24 hours of freezing, the other was resuscitated after freezing for more than two years. The recovered samples were divided into three parts, and they were treated with PBS, NAD and NAM and incubated at 37 °C for 30 min, then the three indexes in the samples were detected. Result After treated with NAD/NAM before freezing, sperm motility and acrosin activity in the samples were increased (P <0.05), but no significant change in DFI was found (P >0.05); After resuscitation, sperm motility and acrosin activity were significantly decreased [(30.5±4.5%) vs (63.3±6.8%)] [(78.2±8.5 µIU) vs (135.0±8.2µIU)] (P <0.05), and DFI was significantly increased [(25.5+1.5%) vs (10.2± 8.3%)] (P <0.05), However, sperm motility in NAD/NAM group was significantly higher than that in control group [(38.4±8.4%)/(36.5± 7.4%) vs (30.5± 4.5%)] (P < 0.05), acrosin activity was significantly higher than that in control group [(90.4± 6.5µIU)/(92.5± 5.8 µIU) vs (78.2±8.5µIU)] (P <0.05), and DFI was significantly lower than that in control group [(16.5±6.8%)/(18.8±8.7%) vs (25.5 ± 7.5%)] (P < 0.05). After NAD/NAM intervention, the sperm motility and acrosin activity of the postresuscitation intervention group were higher than those of the control group [(40.2± 6.3%)/(42.2± 8.8%) vs (31.2± 7.5%)]; [(98.5±7.6µIU)/(93.8±8.2µIU) vs (80.3±5.8µIU)] (P <0.05), but no obvious change in DFI (P >0.05). The sperm motility and acrosin activity of the group freezing more than two years were lower than those of the group freezing 24 h, [(24.5±4.7%) is (33.5±7.4%)]; [(62.4±8.3µIU) ws (83.5±8.3µIU)] (P<0.05), After NAD/NAM intervention, sperm motility and acrosin activity were increased (P <0.05), and this effect was more obvious in cryopreservation group more than two years. Condusion Adding NAD before semen freezing can protect sperm motility, DNA and acrosin activity well, and this effect can last until sperm resuscitation. For long-term frozen sperm, NAD has more significant protective effect. [ABSTRACT FROM AUTHOR]

IntroductionIn the present study, the synergistic protective effect of co-supplementation of glutathione (GSH) with selenium nanoparticles (SeNPs) on the cryopreservation efficiency of bull semen was analyzed.MethodsAfter collection, the ejaculates of Holstein bulls were subsequently diluted with a Tris extender buffer supplemented with different concentrations of SeNPs (0, 1, 2, and 4 μg/ml), followed by semen equilibration at 4°C and assessment of sperm viability and motility. Subsequently, the ejaculates of Holstein bulls were pooled, split into four equal groups, and diluted with a Tris extender buffer supplemented with basic extender (negative control group, NC group), 2 μg/ml SeNPs (SeNPs group), 4 mM GSH (GSH group), and 4 mM GSH plus 2 μg/ml SeNPs (GSH + SeNPs group). After cryopreservation, motility, viability, mitochondrial activity, plasma membrane integrity, acrosome integrity, concentration of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), and ability of frozen-thawed sperm cells to support in vitro embryonic development were evaluated.Results and discussionNo side effect of SeNPs concentrations applied in the current study on the motility and viability of equilibrated bull spermatozoa was found. Meanwhile, supplementation of SeNPs significantly promoted the motility and viability of equilibrated bull spermatozoa. Furthermore, the co-supplementation of GSH with SeNPs effectively protected bull spermatozoa from cryoinjury as expressed by promoting semen motility, viability, mitochondrial activity, plasma membrane integrity, and acrosome integrity. Finally, the enhanced antioxidant capacity and embryonic development potential in the frozen-thawed bull spermatozoa cryopreserved by co-supplementation of GSH with SeNPs further confirmed the synergistic protective effect of co-supplementation of GSH with SeNPs on the cryopreservation of bull semen.

Semen cryopreservation is one common tool for extended genome storage and serves as insurance for declining biodiversity. In the endangered Bornean sun bear, semen was collected with electroejaculation and this is the first attempt at semen cryopreservation for this subspecies. Chilled semen with motility of more than 50 % was extended with Caniplus freezing medium, and cryopreserved with slow freezing protocol. A total of 22 cryopreserved semen straws were evaluated with computer-assisted sperm analysis and conventional semen evaluation tests as well as additional functional tests including evaluation of the acrosome (Rose Bengal Fast Green stain), plasma membrane (hypoosmotic swelling test), and chromatin (toluidine blue stain). Post-thaw semen quality was compromised with poor viability (27.6 ± 11.2 %), motility (8.3 ± 7.1 %), and progressive movement (1.3 ± 2.0 %) but maintained high integrity for the acrosome (71.1 ± 10.8 %), plasma membrane (54.7 ± 9.6 %), and chromatin (89.7 ± 7.6 %). The total post-thaw sperm abnormality was 51.7 ± 10.7 %, predominantly bent tail (28.0 ± 7.6 %) and proximal droplets (20.0 ± 11.8 %). Although this current cryopreservation was not a success, the compromised frozen-thawed semen may be a valuable resource in assisted reproductive technologies such as intracytoplasmic sperm injection. The three additional functional tests demonstrated were simple and affordable and hence are recommended to be part of the routine semen evaluation. Further research is required to develop species-specific cryopreservation protocols and to explore other assisted reproductive technologies in the Bornean sun bear.

Platelet rich plasma (PRP) is extensively used in regenerative medicine. Present work was aimed to investigate the effect of autologous PRP supplementation in semen freezing extender on sperm quality, antioxidant capacity, lipid peroxidation and in vitro fertilizing capacity of frozen-thawed buffalo semen and subsequent embryo developmental competence. Buffalo bulls, n = 8, were used as semen donors. Semen ejaculates were separately divided into four equal parts and extended with autologous PRP 0 (control), 2, 5 and 10% supplemented Tris-based semen extender. Extended semen samples were then cooled to 5 °C for 2h and processed for cryofreezing in French straws. Post-thawed semen samples (37 °C for 30 s) were evaluated for progressive motility (PM), structural membrane integrity (SMI), functional membrane integrity (FMI), total abnormalities (TA), and acrosome integrity (AI). Supernatant from the thawed samples was examined colormetrically for superoxide dismutase activity (SOD), total antioxidant capacity (TAC) and lipid peroxidation profile (MDA). Fertilizing capacity of post-thaw spermatozoa cryofrozen in 5% PRP extender was tested upon fertilization the buffalo oocytes in vitro. Higher (P < 0.05) post-thaw sperm quality (PM, SMI, FMI, AI) in 5% PRP semen extender, whereas TA was lower in control, 2% and 10% concentrations. Five percent PRP supplemented semen extender resulted in greater (P < 0.05) TAC and SOD, and lesser MDA levels compared to other groups. Inseminated buffalo oocytes with sperm cryofrozen in 5% PRP revealed higher fertilization, cleavage and blastocyst rate and lower polyspermy as compared to control. In conclusion, buffalo spermatozoa cryofrozen in autologous PRP supplemented semen extender enhanced cryotolerance and fertilizing potential. • The current work aimed to investigate the effects of autologous PRP supplemented in the freezing extender on the sperm quality. • Progressive motility, structural and functional membrane integrities, total abnormalities, and acrosome integrity were assessed following thawing of semen samples that previously diluted with freezing extender that provided with different concentrations of PRP. • In conclusion, buffalo spermatozoa treated with autologous PRP at pre-freeze, especially with a concentration of 5%, enhances its cryotolerance and fertilizing potential. [ABSTRACT FROM AUTHOR]

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Cryopreservation of bovine semen plays a vital role in accelerating genetic improvement and elite breeding, but it has a detrimental effect on sperm quality, resulting in the decline of the reproductive efficiency. The glycosylation modification of protein has irreplaceable roles in spermatozoa. Herein, the effect of cryopreservation on glycoproteins of bovine spermatozoa has been studied for the first time using a tandem mass tag (TMT)-labeled quantitative glycoproteome. A total of 2598 proteins and 492 glycoproteins were identified, including 83 different expression proteins (DEPs) and 44 different expression glycosylated proteins (DEGPs) between fresh and frozen spermatozoa. Thirty-three DEPs are glycoproteins, which demonstrates that glycoproteins of bovine sperm were seriously affected by cryopreservation. Moreover, the effects include glycoprotein expression, glycosylation modification, and substructure localization for proteins such as glycoproteins TEX101, ACRBP, and IZOMU4. The biologic functions of the 115 changed proteins are mainly involved in sperm capacitation, migration in female genitalia, and sperm–egg interaction. Mostly key regulators were identified to be glycoproteins, which confirms that glycosylated proteins played important roles in bovine sperm. This comprehensive study of sperm glycoproteins helps to unravel the cryoinjury mechanisms, thus implying that glycoprotein protection should be an effective way to improve the quality of frozen sperm. [ABSTRACT FROM AUTHOR]

Background and Aim: The Javan leopard (Panthera pardus melas Cuvier, 1809) is a subspecies of Panthera pardus spp., spread across the African and Asian regions. Information on reproductive aspects is crucial for wild animals, including the Javan leopard. In this study, we aimed to develop electroejaculator (EE) techniques and evaluate cryopreservation success in Javan leopard semen. Materials and Methods: The semen of four adult Javan leopards was collected once a week using EE. Placement of the EE probe in the rectum was performed after ultrasound imaging (ultrasonography) to determine the prostate body location. The semen obtained was then evaluated macroscopically and microscopically. Three Javan leopards were used for cryopreservation. The ejaculate was divided into two parts [i.e., one part diluted with AndroMed® (Minitüb, Tiefenbach, Germany) and the other part with Steridyl® (Minitüb, Tiefenbach, Germany)] at a 1:1 ratio immediately after collection and evaluation. The semen was then packed in a 0.25 mL MiniStraw® (Minitüb, Tiefenbach, Germany) then equilibrated at 4°C for 2 h. After equilibration, the straw was then frozen in liquid nitrogen vapor. Frozen semen was then stored in containers until further evaluation. Results: The results showed that ejaculation response occurred at all levels of stimulation, while erections did not always occur. The fastest ejaculation and erection occurred at the fourth voltage. The macroscopic evaluation showed that the semen volume was 0.80±0.26 mL, cloudy white, pH 7.44±0.14, and with watery semen consistency. The microscopic evaluation showed that the sperm motility was 66.98±0.39%, with sperm viability of 75.6±1.79%. Sperm concentration was 62.17±46.95×106 mL–1 with a total concentration of 42.14±23.51×106 cells. Normal sperm morphology is only 40.72±6.26%. Conclusion: This study concluded that the development of a semen collection technique using an EE preceded by imaging of the EE probe location using ultrasound was effective for the ejaculation of Javan leopards. The characteristics of the semen of the Javan leopard showed moderate semen volume, sperm motility, and viability. Javan leopard showed low sperm concentration and normal sperm morphology.

Caffeine is widely known for its phosphodiesterase enzymes inhibiting, and its powerful reactive oxygen species scavenging property. This study aimed to evaluate the effect of caffeine supplementation @ 0 mM, 1 mM, 3 mM and 5 mM in AndroMed® extender on cryopreservation of Jaffarabadi buffalo semen. Twenty-four semen ejaculates (6/bull) with >70% initial sperm motility were obtained from four mature Jaffarabadi bulls using AV. The ejaculates were split-diluted in an egg-yolk-free AndroMed® extender supplemented with different concentrations of caffeine and were cryopreserved using a standard protocol. The semen samples were evaluated for sperm quality parameters as well as oxidative stress parameters, viz., lipid peroxidation, and Glutathione-S-transferase (GST) enzyme activity in seminal plasma at pre-freeze (after equilibration) and post-thaw stage. The levels of caffeine had a significant effect on all these parameters, except sperm abnormalities, at both pre-freeze and post-thaw stages. Supplementation of caffeine in semen extender at 1 mM and 3 mM concentration showed a significant (p<0.05) increase in post-thawed sperm motility (62.04 ± 0.84, 61.25 ± 1.01%), viability (64.21 ± 0.88, 64.25 ± 0.84%), acrosome integrity (58.08 ± 1.08, 57.30 ± 0.93%) and plasma membrane integrity (52.75 ± 0.89, 52.71 ± 0.74%) and a significant (p<0.05) decrease in oxidative damage as evident by lower lipid peroxidation (MDA 7.62 ± 0.41, 7.80 ± 0.70 µM) and GST enzyme activity (31.15 ± 1.36, 29.54 ± 0.54 nmol CDNB/mL/min) as compared to control and 5 mM caffeine. The study concludes that the post-thaw quality of frozen semen of Jaffarabadi buffalo bulls improves significantly with decreased oxidative stress if the AndroMed® extender is supplemented with 1 and 3 mM concentration of caffeine over control. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) exert an adverse effect on sperm quality during the freezing process. Gamma-oryzanol is an effective antioxidant and has the ability to inhibit lipoperoxidation in various cells. Therefore, this study aims to investigate the effect of gamma-oryzanol supplementation in extender on post-thawed motility and proteomic profiles of swamp buffalo spermatozoa. Each ejaculate of an individual bull was divided into four equal aliquots. Gamma-oryzanol was supplemented at 0 (control), 0.1, 0.25, and 0.5 mM in tris-citrate egg yolk extender. The parameters of sperm motility were evaluated using computer assisted semen analyzer (CASA). The results showed that the progressive motility was significantly higher in 0.5 mM of gamma-oryzanol supplementation group when compared with the control group (p < 0.05), but no significant differences were observed among the treatments. In addition, a proteomic approach was applied to analyze the differentially expressed proteins in post-thawed sperm with or without gamma-oryzanol supplementation in extender. We confirmed that 2-phospho-d-glycerate hydro-lyase (ENO1), glutathione s-transferase mu 1 (GSTM1), phospholipid hydroperoxide glutathione peroxidase (GPX4), outer dense fiber protein 2 (ODF2), tektin-4 (TEKT4), tubulin beta-4B chain (TUBB4B), and ATP synthase subunit beta (ATP5B) were up-regulated in 0.5 mM of gamma-oryzanol supplementation group, which might be associated with the improved post-thawed motility observed in this treatment group. These results demonstrate the beneficial effect of gamma-oryzanol on post-thawed survival of swamp buffalo spermatozoa and help advance the understanding about molecular metabolism of sperm in this species. • 0.5 mM of gamma-oryzanol supplementation in extender enhances the quality of swamp buffalo sperm cryopreservation. • ENO1, GSTM1, GPX4, ODF2, TEKT4, TUBB4B, and ATP5B might be associated with the quality of freeze-thawed swamp buffalo sperm. • These results are beneficial for improving post-thawed sperm and understanding the physiology of swamp buffalo spermatozoa. [ABSTRACT FROM AUTHOR]

In this study, we explore the effects of Poria cocos mushroom polysaccharides (PCPs) on the quality and DNA methylation of the cryopreserved spermatozoa of Shanghai white pigs. A total of 24 ejaculates (three ejaculate samples per boar) from eight Shanghai white pigs were manually collected. The pooled semen was diluted with a based extender supplemented with different concentrations of PCPs (0, 300, 600, 900, 1200, and 1500 μg/mL). Once thawed, the quality of the spermatozoa and their antioxidant function were assessed. In the meantime, the effect of spermatozoa DNA methylation was also analyzed. The results show that compared with the control group, 600 μg/mL of PCPs significantly improves the spermatozoa viability (p < 0.05). The motility and plasma membrane integrity of the frozen–thawed spermatozoa are significantly higher after treatment with 600, 900, and 1200 μg/mL of PCPs compared with the control group (p < 0.05). In comparison with the control group, the percentages of acrosome integrity and mitochondrial activity are significantly enhanced after the application of 600 and 900 μg/mL PCPs (p < 0.05). The reactive oxygen species (ROS), the malondialdehyde (MDA) levels, and the glutathione peroxidase (GSH-Px) activity, in comparison with the control group, are significantly decreased in all groups with PCPs (all p < 0.05). The enzymatic activity of superoxide dismutase (SOD) in spermatozoa is significantly higher in the treatment with 600 μg/mL of PCPs than in the other groups (p < 0.05). As compared with the control group, a significant increase in the catalase (CAT) level is found in the groups with PCPs at 300, 600, 900, and 1200 μg/mL (all p < 0.05). In comparison with the control group, the 5-methylcytosine (5-mC) levels are significantly decreased in all groups with PCPs (all p < 0.05). As a result of these findings, a certain amount of PCPs (600–900 μg/mL) added to the cryodiluent can significantly improve the quality of Shanghai white pig spermatozoa and can also reduce the methylation of spermatozoa DNA caused by cryopreservation. This treatment strategy may establish a foundation for the cryopreservation of semen from pigs.

One of the factors responsible for less pregnancy rates is the use of frozen semen in sheep due to the oxidative stress created by the process. The aim of this experiment was to test the effects of adding coenzyme Q-10 (CoQ10) to the seminal extender on sperm quality and the pregnancy rate of sheep. In this study, ejaculates from eight Dorper rams of reproductive age were used and tested in four treatments: Control (pure BotuBov®), C1 (175 µM of CoQ10), C3 (350 µM of CoQ10), and C7 (700 µM of CoQ10). Samples were collected in triplicate from each animal, and sperm analysis was performed by CASA after thawing at 0 h and 2 h. The samples were also analyzed by flow cytometry for plasma and acrosomal membrane integrity, stability, lipid peroxidation, mitochondrial potential, and superoxide anion production. In total, 198 ewes were inseminated by laparoscopy and divided into two groups: control (n =98) and C7 (n =100). Pregnancy diagnosis was performed at 30 days. Coenzyme Q10 proved to be safe for semen cryopreservation, not altering sperm kinetic values between the groups post-thawing. In flow cytometry, the C1 and C7 groups achieved a better index of plasma membrane integrity and membrane stability (P <0.05). A increased pregnancy rate was observed in C7 (52 %) compared to the control (38 %). In conclusion, coenzyme Q10 assists in the cryopreservation process, protecting the sperm cell and improving pregnancy rates in ewes. • Coenzyme Q10 is an option for cryopreservation of sheep semen. • Coenzyme Q10 improves the pregnancy rate of sheep. • Membrane integrity is increased with Coenzyme Q10 supplementation. [ABSTRACT FROM AUTHOR]

Semen cryobanks are critical for preserving autochthonous and rare breeds. Since sperm cryopreservation has been optimized for commercial breeds, non-commercial ones (often endangered) must be characterized to ensure the germplasm’s viability. This study reports an investigation of the “Asturiana de la Montaña” breed (AM), a valuable Spanish autochthonous cattle breed adapted to the mountainous Atlantic environment. The survey included cryopreserved semen doses from 40 bulls stored at the Principado de Asturias Germplasm Bank. Data were obtained from the routine fresh semen analysis, CASA (motility), and flow cytometry analyses of fresh and post-thawing semen, and the 56-day non-return-rate (NRR) in heifers and cows (all results as 1st and 3rd quartiles). Fresh samples (artificial vagina) were within the normal range for cattle (4–6 mL, 5–10 × 109/mL; mass motility 5). Post-thawing results showed motility below typical for commercial breeds (total motility 26–43%, progressive 14–28%), with higher values for viability (47–62%). Insemination results showed a good performance for this breed (NRR: 47–56%; higher for heifers). Sperm volume increased with age, with little or no effects on sperm quality. Few associations were found between post-thawing quality or freezability and NRR, LIN being the variable more strongly associated (positively). The AM semen bank shows a good prospect for preserving and disseminating the genetics of this breed. This survey indicates that dedicated research is needed to adapt freezing protocols to this breed, optimizing post-thawing results.

Semen cryopreservation is crucial maintain genetic diversity of avian species. Little is known about suitable extenders for post-thaw survival of spermatozoa from Thai red junglefowl (Gallus gallus gallus). Therefore, the present study aimed to compare the suitability of the modified Thai red junglefowl extender (TRJFE) for cryopreservation of Thai red junglefowl spermatozoa with other extenders, including the Schramm extender, the red junglefowl extender (RFE), and the HS1 extender, in terms of sperm viability, motility, and fertility. First, the effects of adding 6% and 9% (v/v) N , N -dimethylacetamide, N , N -dimethylformamide (DMF), or N -methyl acetamide to these extenders on the post-thaw sperm quality of pooled ejaculates from 25 male fowls. The viability of thawed sperm was assessed by using nigrosin–eosin staining and sperm motility was determined by using computer-assisted sperm analysis. Fertility was assessed by inseminating 144 laying hens. The TRJFE +6% DMF combination significantly improved post-thaw viability (64.88 ± 0.51%) and motility (68.58 ± 1.13%) of Thai red junglefowl sperm, and the semen had the highest fertility (60.97 ± 0.72%). The findings suggest that TRJFE +6% DMF is a suitable freezing medium to conserve Thai red jungle fowl genetic resources. [ABSTRACT FROM AUTHOR]