Semen Cryopreservation Research Articles

Association between different soy lecithin-based extenders and freezing rates in ram semen cryopreservation

The aim of the study was to evaluate the effect of the association between glycine-milk (GM) based extenders made with different concentrations of soy lecithin (SL) and freezing rates (FR) on semen quality after thawing. Pooled semen from rams (n = 12) were diluted in GM extenders with 20% egg yolk (EY–20%) or with different concentrations of SL: 0.5% (SL–0.5%), 1.0% (SL–1.0%), and 2.0% (SL–2.0%). The diluted semen (150 ×106 spermatozoa/0.25 mL) was frozen at three FR of − 10, − 20, and − 60 °C/min, and subsequently thawed and analyzed. Results revealed that EY–20% and SL–2.0% had better kinetic parameters, and showed higher proportions of viable, non-apoptotic, plasma-membrane-intact spermatozoa (A-/PI-) and non-capacitated spermatozoa (F), and had lower acrosome-reacted spermatozoa (AR) in the EY–20% and satisfactory values for SL-2.0% compared to SL–0.5% and SL–1.0% (P < 0.05). The FR at − 20 and − 60 °C/min maintained higher A-/PI- and viable spermatozoa compared to − 10 °C/min. The combination EY–20% and − 60 °C/min showed the highest A-/PI- and F (P < 0.05) and the lowest AR, and it did not differ from the combinations EY–20% at − 20 °C/min and SL–2.0% at − 20 °C/min (P > 0.05). In conclusion, the combination EY–20% and − 60 °C/min, showed the best cryoprotective effects on ram spermatozoa. Changes in spermatozoa after thawing were related to the use of the type of extender, the amounts of the same compound in the extender, and the freezing rates to which they were subjected.

Tinospora cordifolia modulates the seminal parameters, leakage of intracellular enzymes and seminal antioxidants in equilibrated and cryopreserved semen of Sahiwal bulls.

The present study was undertaken to assess the effects of stem extract of Tinospora cordifolia (Giloy or Guduchi) in the semen extender on seminal parameters, leakage of intracellular enzymes and antioxidants in semen of Sahiwal bull. A total of 48 ejaculates from four bulls were selected for the study. Spermatozoa of 25×106 were incubated in 100, 300 and 500 μg of stem extract of Guduchi as Gr II, III and IV, respectively and pre-freeze and post-thaw semen samples were analysed for seminal parameters (motility, viability, total sperm abnormality; TSA, plasma membrane integrity; PMI, and acrosomal integrity; AcI), intracellular enzymes (aspartate aminotransferase; AST and lactate dehydrogenase; LDH) and seminal antioxidants (superoxide dismutase; SOD and catalase) in comparison with an untreated control group (Gr I). The results revealed that stem extract treated semen had significantly (p<0.05) higher motility, viability, PMI, AcI, SOD and catalase and had significantly (p<0.05) lower TSA, AST and LDH compared to those in untreated control group at pre-freeze and post-thaw stages. Semen treated with 100 μg stem extract /25×106 spermatozoa had significantly (p<0.05) higher motility, viability, PMI, AcI, SOD and catalase and had significantly (p<0.05) lower TSA, AST and LDH compared to those in control, 300, and 500 μg treated groups at pre-freeze and post-thaw stages. Further, these seminal parameters and antioxidants were showing decreasing trend and TSA and leakage of intra-cellular enzymes were showing increasing trend from Gr II to Gr IV at pre-freeze and post-thaw stages. Thus, 100 μg/25×106 spermatozoa were optimum or suitable dose for cryopreservation of Sahiwal bull semen. The study concluded that T. cordifolia stem extract 100 μg/25×106 spermatozoa in the semen extender can be effectively utilized to reduce the oxidative stress and improve the pre-freeze and post-thaw seminal parameters in Sahiwal bull. However, further studies on effects of different concentrations of stem extract on in-vitro or in-vivo fertility trials are to be conducted to assess the impact of the stem extract supplementation in the semen extender on field pregnancy outcome in bovine species.

MP79-08 EFFECT OF HORMONE THERAPY ON SEMEN ANALYSIS PARAMETERS IN ADOLESCENT TRANSGENDER FEMALES

You have accessJournal of UrologyCME1 Apr 2023MP79-08 EFFECT OF HORMONE THERAPY ON SEMEN ANALYSIS PARAMETERS IN ADOLESCENT TRANSGENDER FEMALES Sai Allu, Sreeya Komaravolu, Mark Sigman, Gabriella Avellino, and Sarah Moore Sai AlluSai Allu More articles by this author , Sreeya KomaravoluSreeya Komaravolu More articles by this author , Mark SigmanMark Sigman More articles by this author , Gabriella AvellinoGabriella Avellino More articles by this author , and Sarah MooreSarah Moore More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003356.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Feminizing transgender patients represent an important urological patient population with unique healthcare needs. Although not every patient chooses to cryopreserve, everyone should be given the option. Hormone therapy (HT) is often started prior to proceeding with surgical orchiectomy. Research has suggested that transgender females on HT have reduced semen analysis (SA) parameters compared to cisgender patients, including reduced sperm concentration and motility. [1,2] There is limited information regarding fertility preservation by transgender patients on HT. The goal of this study is to report cryopreservation outcomes by adolescent feminizing transgender patients who are currently on HT. METHODS: We retrospectively reviewed charts of donors with our Men’s Health Center for direct donations to the sperm bank between 01/2011 to 07/2022. Inclusion criteria included adolescent age (16–24 years old) and the use of HT within three months of cryopreservation. Patient data collected included patient demographics, type of hormone therapy, and SA parameters. RESULTS: Five transgender patients were found who were on HT within three months prior to their semen cryopreservation and analysis. Their median age was 18 (range 16–22). Three patients were on spironolactone, one patient was on estradiol, and one patient was on a combination of both medications. All four patients on single-medication HT had normal concentration and motility on their semen analysis, as defined by the WHO Laboratory Manual for the Examination and Process of Human Semen (Table 1). Three of four patients on single HT had normal volume. The patient on combination HT demonstrated lowered overall SA parameters. CONCLUSIONS: This is the largest cohort of adolescent feminizing transgender patients on hormone therapy in which fertility preservation has been described. Semen cryopreservation could be a viable method for fertility preservation among this patient population who have already initiated hormonal therapies, specifically spironolactone or estradiol. Patients on single HT appear to have improved semen analysis parameters compared to patients on combination HT. Future studies are needed to further characterize fertility preservation with specific information on duration and dosage of HT, as well as outcomes of cryopreservation. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1144 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sai Allu More articles by this author Sreeya Komaravolu More articles by this author Mark Sigman More articles by this author Gabriella Avellino More articles by this author Sarah Moore More articles by this author Expand All Advertisement PDF downloadLoading ...

MP01-18 DECLINE IN SPERM FREEZE-THAW RECOVERY RATE WITH DELAYED CRYOPRESERVATION

You have accessJournal of UrologyCME1 Apr 2023MP01-18 DECLINE IN SPERM FREEZE-THAW RECOVERY RATE WITH DELAYED CRYOPRESERVATION Andre Belarmino, Daniel Colin, Stacey Kenfield, Stanton Honig, James Dupree, Mary Samplaski, Dolores Lamb, Jerrine Morris, and James Smith Andre BelarminoAndre Belarmino More articles by this author , Daniel ColinDaniel Colin More articles by this author , Stacey KenfieldStacey Kenfield More articles by this author , Stanton HonigStanton Honig More articles by this author , James DupreeJames Dupree More articles by this author , Mary SamplaskiMary Samplaski More articles by this author , Dolores LambDolores Lamb More articles by this author , Jerrine MorrisJerrine Morris More articles by this author , and James SmithJames Smith More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Sperm cryopreservation requires in-person visits to produce an ejaculate and immediately freeze the sample. Recently, companies began offering services for mail-in semen analysis (SA) testing and cryopreservation. However, the effects of delayed cryopreservation associated with shipping times were generally understudied. Our laboratory has continued to optimize our cryopreservation technique. This study aims to define the effect of delayed cryopreservation on the recovered total motile count (TMC) compared to standard immediate cryopreservation, using our improved methods. METHODS: Fresh semen samples were provided from healthy volunteers. Samples were diluted with 5 mL of a proprietary preservation solution, mixed, and allowed to liquify for 30 minutes. Samples were then analyzed per World Health Organization (WHO) guidelines V5 and those with abnormal concentration, volume, or motility were excluded. Samples were then divided into 3 aliquots. Aliquot #1, T0 was immediately frozen with equal part cryoprotectant. Aliquot #2, T24 was left at room temperature for 24hrs, analyzed on the SQA (sperm quality analyzer) and then frozen with cryoprotectant. Aliquot #3, T48 was left at room temperature for 48hrs, analyzed on the SQA, and then frozen with cryoprotectant. 24hrs after the third aliquot was frozen, all aliquots were thawed in an incubator at 37C for 10 minutes and analyzed on the SQA. RESULTS: Eight adult men provided a total of 26 semen samples. Baseline SA values were: volume 3.4 ml±1.2, concentration 128.2±48.7 × 106, motility 49%±7%; TMC 211±109 million. A decline in % motility was observed after specimens sat at room temperature, as well as after freeze-thaw in most individuals. The average loss from freeze/thaw in TMC at T0 was 44.0%, at T24 was 47% relative to T24 pre-freeze, and at T48 was 65% relative to T48 pre-freeze (p=0.046) (Figure 1). CONCLUSIONS: This analysis reveals a superior freeze-thaw protocol compared to our prior methods, as we continue to refine our process, and follow best practice guidelines of experts in the field. Further investigation is needed to improve the methods needed for shipping with the goal of improving the freeze-thaw recovery rate with delayed cryopreservation. Source of Funding: Fellow Health Inc. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e9 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Andre Belarmino More articles by this author Daniel Colin More articles by this author Stacey Kenfield More articles by this author Stanton Honig More articles by this author James Dupree More articles by this author Mary Samplaski More articles by this author Dolores Lamb More articles by this author Jerrine Morris More articles by this author James Smith More articles by this author Expand All Advertisement PDF downloadLoading ...

Carboxymethylchitosan with medium-molecular-weight affects kinectics and acrosome of stallion sperm after freezing/thawing

Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were filled in straws (0.5 mL) and submitted to freezing and storage at -196°C. Thawing was performed at 37°C/30 s, and the samples of each group were analyzed for kinetics, plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential . The addition of 1.5 and 3 mg/mL CQm showed lower values (P < .05) of total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and wobble (WOB), comparing to control group. Besides, it was observed lower (P < .05) percentages of sperm with intact acrosomes in the group treated with 3 mg/mL of CQm than control group. In conclusion, high concentration of medium-molecular-weight carboxymethylchitosan to freezing diluent damages kinematic and acrosome of stallion sperm after freezing/thawing.

ARTIFICIAL INSEMINATION IN GOAT: A NEW PROSPECT FOR SCIENTIFIC GOAT BREEDING

In India major portion of goat farming is extensive and breeding system follows uncontrolled natural mating. For better breeding management there should be sufficient good quality stud bucks. But it is very unfortunate that due to lack of breeding buck more than 30% estrus does remain without service. In order to check the negative selection and for proper management of breeding policy, cryopreservation of superior native buck semen and artificial insemination is the only way. Artificial insemination (AI) is a part of assisted reproductive technology (ART) providing augmentation of genetic make-up in individuals. Artificial insemination in sheep and goat was first described detail in 1987. Globally 0.5 million AIs are performed annually in case of goat which is very less than other animals. Research in the area of cryopreservation of goat semen and artificial insemination has been limited to few research institutes and AI is least practiced in field condition. Intra-cervical insemination is widely practiced in case of does with successful results.

Raptors bred in captivity: semen characteristics and assisted reproduction outcome in goshawk (Accipiter gentilis).

Three sexually mature goshawks reared in captivity and imprinted on humans to express reproductive behavior according to the cooperative method were studied for three consecutive breeding seasons to assess the quality of their sperm. The following parameters were analyzed: ejaculate volume and sperm concentration, motility, viability, and morphology. Ejaculate volume, sperm concentration and motility fluctuated along the reproductive season, revealing the greatest quality of the reproductive material at full springtime (i.e., April). Motility of the sperm collected in March strongly reduced with age, contrary to samples collected in April or May. Sperm viability was not influenced by either age or month of collection within each season. Ultrastructural investigations provided information on normal sperm morphology for the first time in this species. The morphological categories of sperm defects in fresh semen, present at low percentages, are also described. Functional analyses (perivitelline membrane assay and artificial inseminations) confirmed the good quality of the semen obtained using the cooperative method. The reported data provide the basis for further studies aimed at developing protocols to improve the outcome of artificial insemination and semen cryopreservation in the goshawk as well as other bird of prey species.

Freezability and Fertility Rates of Stallion Semen Supplemented With Trehalose in Lactose Extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. One of the reason for this diminished quality is osmotic stress that spermatozoa experiences during freezing and thawing process. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze–thawing process. Semen samples were collected from six Marwari breed stallions, divided into three different treatments in a final concentration of 150 × 106 sperm/mL by using Lactose based extender containing 0, 50, and 150 mM of trehalose then subjected to cryopreservation after equilibration. Sperm motility, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, DNA integrity and oxidative stress related parameters of the stallion spermatozoa were analyzed at fresh, prefreeze and post thaw stages. Thirty (30) reproductively healthy mares were inseminated with frozen-thawed semen either supplemented with (treatment) or without (control) trehalose to evaluate the field fertility. Results of the current study indicated that, the extender containing 50 mM trehalose has enhanced the functional plasma membrane, acrosomal, DNA integrities and augmented the mitochondrial membrane potential. Trehalose supplementation to the semen extender not only ameliorated the semen quality parameters, but also protected the stallion sperm from oxidative stress by reducing the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO). The inclusion of 50 mM trehalose in semen extender resulted in significantly (P < .05) increased post-thaw progressive motility and viability compared to the control group. Mares inseminated with frozen-thawed semen supplemented with 50 mM trehalose tended to have better pregnancy rates than controls (non-significant [P < .05]) although a larger fertility trial is required to determine if this effect reaches the level of significance. In conclusion, addition of 50 mM trehalose yielded in better quality stallion semen after cooling and post-thawing in terms of reducing the oxidative stress and enhancing the motility, integrities of acrosome, plasma membrane, mitochondrial potential and DNA.

Biomaterials for Testicular Bioengineering: How far have we come and where do we have to go?

Traditional therapeutic interventions aim to restore male fertile potential or preserve sperm viability in severe cases, such as semen cryopreservation, testicular tissue, germ cell transplantation and testicular graft. However, these techniques demonstrate several methodological, clinical, and biological limitations, that impact in their results. In this scenario, reproductive medicine has sought biotechnological alternatives applied for infertility treatment, or to improve gamete preservation and thus increase reproductive rates in vitro and in vivo. One of the main approaches employed is the biomimetic testicular tissue reconstruction, which uses tissue-engineering principles and methodologies. This strategy pursues to mimic the testicular microenvironment, simulating physiological conditions. Such approach allows male gametes maintenance in culture or produce viable grafts that can be transplanted and restore reproductive functions. In this context, the application of several biomaterials have been proposed to be used in artificial biological systems. From synthetic polymers to decellularized matrixes, each biomaterial has advantages and disadvantages regarding its application in cell culture and tissue reconstruction. Therefore, the present review aims to list the progress that has been made and the continued challenges facing testicular regenerative medicine and the preservation of male reproductive capacity, based on the development of tissue bioengineering approaches for testicular tissue microenvironment reconstruction.

Antioxidant additive melatonin in tris-based egg yolk extender improves post-thaw sperm attributes in Hariana bull

In the study, melatonin, a known antioxidant pineal peptide was used as an additive in the tris-egg yolk glycerol-based semen extender in Hariana bull semen and post-thaw sperm characters were evaluated. In the study, Group I was a control without melatonin; and Group II, III, and IV were having 0.5 mM, 1 mM, and 2 mM melatonin/80 × 106 spermatozoa, respectively were treatment groups. Thirty-two semen ejaculates from 4 Hariana bulls were processed for freezing and post-thaw sperm characteristics were evaluated. Sperm motility, velocity, the viability with intact membrane, and total antioxidant capacity were markedly (P < 0.05) improved in Group IV compared to all other groups. The lipid peroxidation and total protein carbonylation were substantially (P < 0.05) decreased in Group IV compared to all other groups. The population of cryocapacitated, acrosome-reacted, and apoptotic-like spermatozoa were significantly (P < 0.05) decreased in Group IV. Further, the relative band intensity of 74 kDa protein and percent of spermatozoa showing positive immune reactivity to tyrosine-phosphorylated proteins was decreased in Group IV. The progesterone-receptor ligand binding, in vitro capacitation response, and Vanguard distance were markedly (P < 0.05) improved in Group IV. In summary- Group IV having 2 mM melatonin was found to be optimal in providing cryoprotective effects to Hariana bull spermatozoa after freezing-thawing and can be suitably used as a semen additive during semen cryopreservation.

Assisted reproductive technologies (ARTs) in Mithun (Bos frontalis): What progress has been made so far? An overview.

Mithun, a unique bovine species, endemic to parts of North East India and plays an important role in the socioeconomic, cultural and religious fabrics of the local tribal population. To date, Mithuns are reared in a traditional free-range system by communities and increased deforestation, agricultural commercialization, disease outbreaks and indiscriminate slaughtering of elite Mithun for table purposes have significantly decreased its habitat and the elite Mithun population. Greater genetic gain is achieved with the implementation and effective use of assisted reproductive technologies (ARTs); however, presently it is limited to organized Mithun farms. At a slow pace, Mithun farmers are adopting semi-intensive rearing systems and interest in the use of ARTs is gradually escalating in Mithun husbandry. This article reviews the current status of ARTs such as semen collection and cryopreservation, estrus synchronization and timed artificial insemination (TAI), multiple ovulation and embryo transfer and in vitro embryo production and future perspectives in Mithun. Mithun semen collection and cryopreservation have been standardized, and estrus synchronization and TAI are suitable technologies that can be easily implemented under field conditions in near future. The establishment of an open nucleus-breeding system under community participatory mode along with the introduction of the ARTs is an alternative to the traditional breeding system for rapid genetic improvement of Mithun. Finally, the review considers the potential benefits of ARTs in Mithun and future research should include the use of these ARTs which will provide additional opportunities for improved breeding regimens in Mithun.

Extension of the equilibration period up to 24 h maintains the post-thawing quality of Holstein bull semen frozen with OPTIXcell®

Semen cryopreservation in bovine livestock is well established, but logistics often require deviations from standard protocols. Extending the equilibration time to the following day is convenient in many situations. To improve our knowledge of the effects of this modification, we studied the post-thawing and post-incubation (4 h, 38 °C) sperm quality after freezing with 4 or 24-h extension in the OPTIXcell extender by using an ample panel of analyses: CASA for motility; flow cytometry for viability, physiology, oxidative stress, and chromatin parameters (DNA fragmentation, chromatin compaction, and thiol groups status); and spectrometry for malondialdehyde production. Semen was obtained from 12 Holstein bulls. The 24-h equilibration time showed few significant effects, with only a tiny decrease in progressive motility and a positive impact on chromatin structure. The incubation removed some of these effects, with the pattern for chromatin compaction remaining the same. No detrimental oxidative stress or increase in apoptotic or capacitation markers was detected. Additionally, the individual bull interacted with the effects of the incubation and the equilibration, especially regarding the chromatin status. Whereas this interaction did not critically affect sperm quality, it could be relevant in practice. Bull fertility as non-return rates (NRR56) was associated with some sperm parameters (especially with an improved chromatin structure) but not in the 4-h post-thawing analysis. Our study supports that extending the equilibration time by at least 24-h is feasible for bull semen freezing with the OPTIXcell extender.

The Quality of Frozen Friesian Holstein Semen after Long-Term Storage

Semen cryopreservation is the long-term storage at very low temperatures in liquid nitrogen for future use. This study investigates the quality of frozen Friesian Holstein (FH) semen after long-term storage. Samples of FH semen stored for 25, 20, 15, 10, 5, and 1 years were collected from one of the national centers for artificial inseminations. Frozen semen was stored in containers with liquid nitrogen at -196 ᵒC in a room with a temperature of 20 ᵒC The variables used after thawing were sperm motility, viability, and abnormalities, as well as plasma membrane integrity (IPM) using computer-assisted sperm analysis (CASA), eosin-nigrosine staining, and hypoosmotic swelling (HOS) test, respectively. Data were analyzed by one-way ANOVA and expressed as mean ± SEM. The result showed no significant differences in sperm viability, abnormalities, and IPM. Furthermore, sperm motility was >40%, consistent with the Indonesian standard for frozen bovine semen. CASA analysis showed that all variables of the motility pattern have no significant difference, except linearity (LIN). The Lin of sperm was lower in frozen semen after one and five years than after 20 and 25 years of storage. The overall quality of semen after 25, 20, 15, 10, 5, and 1 years of storage met the standard and was suitable for artificial insemination.

Profiling of differentially expressed proteins between fresh and frozen-thawed Duroc boar semen using ProteinChip CM10.

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.

Cryopreservation of Human Spermatozoa: Functional, Molecular and Clinical Aspects.

Cryopreservation is an expanding strategy to allow not only fertility preservation for individuals who need such procedures because of gonadotoxic treatments, active duty in dangerous occupations or social reasons and gamete donation for couples where conception is denied, but also for animal breeding and preservation of endangered animal species. Despite the improvement in semen cryopreservation techniques and the worldwide expansion of semen banks, damage to spermatozoa and the consequent impairment of its functions still remain unsolved problems, conditioning the choice of the technique in assisted reproduction procedures. Although many studies have attempted to find solutions to limit sperm damage following cryopreservation and identify possible markers of damage susceptibility, active research in this field is still required in order to optimize the process. Here, we review the available evidence regarding structural, molecular and functional damage occurring in cryopreserved human spermatozoa and the possible strategies to prevent it and optimize the procedures. Finally, we review the results on assisted reproduction technique (ARTs) outcomes following the use of cryopreserved spermatozoa.

Egg yolk plasma enriched with β-carotene through the diet of laying hens and adding it to the extender improves the quality of frozen semen in Arabic stallions.

Equine semen cryopreservation is one of the major procedures for the genetic conservation of rare and endangered genotypes. The current study was conducted to evaluate the effect of egg yolk plasma (EYP) enriched with β-carotene as an antioxidant supplement on INRA-96 extender regarding freezing Arabic stallion sperm. For this purpose, β-carotene various concentrations were utilized as a supplementary ingredient in formulating the diets of laying hens. Birds were randomly divided into four groups, fed with 0 (control), 500, 1000 and 2000 mg/kg in a supplemented diet with β-carotene. Subsequently, various variants of enriched extender (INRA-96 + 2.5% glycerol [G]) were gained by adding 2% EYP from four treatment groups. The sperm characteristics, including motility, viability, morphology, plasma membrane integrity (HOS test), lipid peroxidation (MDA) and DNA fragmentation, were evaluated after thawing. According to the results obtained in this study, the addition of EYP from T2 and T4 (500 and 2000 mg/kg of β-carotene in hens' diet) to the extender (INRA-96 + 2.5% G) leads to an increase in total motility (50.50% and 49.49%, respectively), progressive motility (32.6% and 31.8%, respectively), viability (68.7% and 66.1%, respectively) and plasma membrane integrity (57.7% and 50.6%, respectively). Moreover, lipid peroxidation (1.3 and 1.4nmol/mL, respectively) and DNA fragmentation (8.6% and 9.9%, respectively) were diminished using the mentioned treatments. However, sperm morphology was not affected by the treatments. In the current study, we concluded that the optimal concentration of β-carotene in the laying hen's diet (500 mg/kg) could reveal the best results about sperm quality. So, EYP enriched with β-carotene acts as a valuable natural and safe supplementary material that could be exploited for enhancing stallion sperm quality in cryopreservation conditions.

COMPARISON BETWEEN RAPID AND SLOW CRYOPRESERVATION PROTOCOLS FOR RAM SEMEN

Information regarding adequate freezing protocols of ram semen considering freezing distance and time during cryopreservation has not been adequately reported. Therefore, this study aimed to compare two freezing protocols for Najdi ram's semen. In the rapid freezing protocol, the straws were frozen at 5 cm over the surface of liquid nitrogen for 15 minutes. While in the slow freezing protocol, the straws were frozen at 8 cm over the surface of liquid nitrogen for 20 minutes. The semen was collected from five rams and extended with tris egg yolk glycerol cryodiluent. The extended semen was chilled slowly to 50C within two hours and equilibrated for two hours before being frozen on the liquid nitrogen vapor and cryopreserved at -1960C. There was no significant (P &gt;0.005) effect of the freezing protocol on the sperm’s total motility, plasma membrane integrity, DNA integrity, and abnormalities. However, the vitality, fast progressive motility, straight-line velocity, average pathway velocity, linearity, and wobble were significantly higher in rapid freezing than in slow freezing protocol. In conclusion, cryopreservation of ram semen using rapid freezing (5 cm for 15 mins) protocol was better than slow freezing (8 cm for 20 mins) protocol regarding post-thawing semen quality. Key words: freezing protocol; cryopreservation; egg yolk; ram; semen

Seasonality does not influence cortisol or testosterone production, or seminal quality of stallions located at low latitudes

The effects of seasonality on the reproduction of stallions vary based on the latitude. Although previous studies have shown the influence of seasonality in raw semen quality in south-eastern Brazil, data regarding the influence of seasonality in cooled and frozen stored semen in Brazil is limited. Therefore, in this study, we have analysed if seasonality influences the hormone production (i.e., cortisol and testosterone), spermatogenesis, and quality of fresh, cooled, and frozen semen of stallions in central Brazil, and established the season most suitable for semen cryopreservation in a latitude of 15°S. Ten stallions were followed-up for one year, which was divided into two seasons, namely, drought, and rainy. Fresh, cooled, and frozen-thawed semen samples were assessed using CASA and flow cytometry. Additionally, the temperature and humidity index (THI) was calculated to determine the thermal stress. Although the THI varied between the two seasons, no thermal stress was observed throughout the year, nor were there differences in the physiological parameters of the stallions or plasma cortisol or testosterone levels. Furthermore, differences were not detected in total and progressive motility, sperm capacitation, and sperm membrane integrity, as well as in the number of live sperm with intact acrosomes and high mitochondrial membrane potential, between the two seasons in the fresh and frozen-thawed semen. Our data suggest that semen can be effectively collected and cryopreserved throughout the year within central regions of Brazil.

Glutathione and selenium nanoparticles have a synergistic protective effect during cryopreservation of bull semen.

In 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. After 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. No 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.

Chicken Sperm Cryopreservation: Review of Techniques, Freezing Damage, and Freezability Mechanisms

Ex situ preservation is an important method in the preservation of chickens, and cryopreservation of semen is the only method for gamete preservation at present. During the last two decades, many studies have been performed to develop standard chicken semen cryopreservation technology and achieve great progress. Many attempts and methods were investigated to adapt subspecies or different breeds. In this paper, we firstly reviewed the main factors affecting cryopreservation of chicken sperm, including the unique structure and characteristics of the spermatozoa. Secondly, the studies on key points of the chicken sperm cryopreservation technology, including semen dilution, cryoprotectants, equilibration time, packaging types, and freezing and thawing rates were summarized to generate the optimal parameters. Then, the mechanism underlying freezing damage and freezability revealed by recent omics methods relevant to the efficiency of cryopreservation were discussed. This review will provide relevant reference for the future investigation of poultry semen cryopreservation technology.