Yolk Buffer Research Articles

P-073 Microfluidic sperm selection yields competent spermatozoa with higher genomic integrity and cryostress tolerance

Abstract Study question Can microfluidic sperm sperm selection (MFSS) contribute to isolating spermatozoa with higher genomic integrity and cryostress resistance, while retaining embryo developmental competence? Summary answer In comparison to conventional density gradient (DG), MFSS yielded a higher proportion of motile spermatozoa coupled with enhanced genomic integrity and resistance to cryostress. What is known already The cryopreservation of human spermatozoa has long been a cornerstone in reproductive treatments, serving as a pivotal method for preserving male fertility for several decades. Despite the unlimited storage time of cryopreserved semen specimens, the thawing process inevitably diminishes the number of viable spermatozoa. The implementation of a technique capable of enhancing the motility of spermatozoa cryopreserved would yield a higher proportion of gametes with higher kinetics and genomic integrity at thawing. Study design, size, duration Since 2017, 183 men underwent cryopreservation utilizing MFSS. Post-processing semen parameters, including Sperm Chromatin Fragmentation(SCF), and post-thaw motility were compared with a DG control(n = 1979). Additionally, post-thaw parameters for 30 men who had previously employed DG and subsequently adopted MFSS for semen cryopreservation were included. In 12 specimens, a paired analysis was carried out on a same ejaculate. Moreover, we explored pregnancy outcomes with MFSS-selected specimens in some men that underwent ART. Participants/materials, setting, methods Semen parameters were analyzed, and DG was performed according to WHO 2021 guidelines. MFSS was carried out following manufacture’s protocol (ZyMōt® Multi 850µL). Cryoprotectant (Test Yolk Buffer) was added to the final sample before being immersed in liquid nitrogen. A minute aliquot of the processed sample, cryopreserved separately, was thawed for post-thaw motility. TUNEL assay was used to measure SCF (≤ 15% normal threshold). Main results and the role of chance In this study, 1979 men (40.0±7y) underwent DG prior to cryopreservation, resulting in a 44.3±25x106/mL concentration, 88.2±4% motility, and 2.9±1% normal morphology, serving as control. The remaining 183 men of a similar age underwent MFSS prior to cryopreservation. Despite a lower concentration at 26.1±15x106/mL(P < 0.001), MFSS resulted in a higher post-processing motility(97.8±2%, P < 0.001) and normal morphology(3.4±1%, P < 0.001). Post-thaw analysis revealed a remarkably higher post-thaw motility in MFSS (51.2±7% vs 43.6%±4%,P < 0.001). SCF averaged 17%(raw), 11%(DG) and 2%(MFSS,P<0.001). To confirm research hypothesis, a pilot study was done in 12 men (37.3±3y,3.0±2d abstinence) with their semen specimen equally aliquoted for DG or MFSS and cryopreserved. The post-thaw motility increased from 45.4±8%(DG) to 61.3±13%(MFSS,P<0.001). Subsequently, in men(n = 30, 40.8±6y, 3.0±2d abstinence) who underwent semen cryopreservation previously by DG and subsequent by MFSS, a similar trend was observed with a higher post-thaw motility (53.2±6% vs 43.2%±6%,P < 0.001). Among the 36 patients who utilized DG cryopreserved specimen for reproductive purposes, 11/19 (57.9%) patients achieved pregnancy in ICSI cycles. while 5/17 (29.4%) achieved pregnancy in an IUI cycle, Among the 18 couples who used MFSS cryopreserved specimen for ICSI, a pregnancy rate was reported (8/18,44.4%). A IUI pregnancy rate was also reported (4/18,22.2%) using MFSS cryopreserved specimen. Limitations, reasons for caution While MFSS has proven to increase post-thaw survival rate and maintain a lower SCF, it did not necessarily trend in a higher pregnancy in IUI cycles, possibly due to the borderline elevated SCF. This finding needs to be confirmed in a larger population. Wider implications of the findings In reproductive medicine, we strive to identify new techniques aimed at providing more competent gametes. MFSS was capable of isolating the most motile potion of spermatozoa with a higher genomic integrity. The utilization of MFSS for specimens to be cryopreserved appears promising in generating higher post-thaw recovery of motile spermatozoa. Trial registration number not applicable

Tip-microVapour Fast Freezing: A novel easy method for cryopreserving severe oligozoospermic samples.

Sperm cryopreservation is an important procedure for oligozoospermic subjects at risk of azoospermia and after surgical recovery of spermatozoa in non-obstructive azoospermic men. Conventional procedures for sperm cryopreservation might be, however, not suitable for samples with a very low sperm number. In this pilot study, we investigated the recoveries of sperm motility and viability in severe oligozoospermic subjects (n=39) after cryopreservation with a tip-microVapour Fast Freezing, a procedure previously developed by our group for men with good semen quality. Sperm DNA fragmentation was also evaluated in a second group of oligozoospermic samples (n=16). We used a Vapour Fast Freezing procedure using 10μL tips as carrier, and Test Yolk Buffer as freezing medium (tip-microVapour Fast Freezing). In a subset of samples (n=22), we compared recovery of motility and viability as obtained with tip-microVapour Fast Freezing and with a Vapour Fast Freezing procedure using 500μL straws. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test. We found a recovery rate (median [interquartile range]) of 0.29 (0.13-0.41) for progressive motility, 0.30 (0.21-0.52) for total motility and 0.48 (0.29-0.60) for viability. Interestingly, we observed that samples with the poorest motility were apparently less damaged by freezing/thawing. In a subset of samples (n=22), we directly compared values of viability, progressive motility and total motility by freezing/thawing with tip-microVapour Fast Freezing and Vapour Fast Freezing conducted with 500μL straws. We found much better values of all sperm parameters in samples after freezing/thawing with tip-microVapour Fast Freezing than with Vapour Fast Freezing in 500μL straws: that is, progressive motility: 7.00 (3.00-8.50)% versus 2.00 (0.00-4.25)%, p<0.001; total motility: 12.00 (8.00-16.25)% versus 6.50 (1.00-9.25)%, p<0.001; viability: 29.75 (23.75-45.25) versus 22.50 (13.75-28.13), p<0.001, respectively. In the second group of oligozoospermic samples, we found that tip-microVapour Fast Freezing produced lower levels of sperm DNA fragmentation than straws (33.00 [19.75-36.00]% vs. 36.00 [22.75-41.87]%, p<0.001). Tip-microVapour Fast Freezing appears to be a very promising method to cryopreserve semen samples from severe oligozoospermic patients.

Addition of Sugarcane Juice to Tris Egg Yolk Buffer Diluent Improves the Quality of the Stored Diluted Semen of Kacang Goats at 5°C

Addition of Sugarcane Juice to Tris Egg Yolk Buffer Diluent Improves the Quality of the Stored Diluted Semen of Kacang Goats at 5°C

Cryopreservation of Sperm from an Endangered Snake with Tests of Post-Thaw Incubation in Caffeine.

Simple SummaryCryopreservation of sperm from reptiles to aid the recovery of endangered species continues to be a challenge. In this study, we tested the cryoperformance of a cryoprotective agent (CPA) mixture to cryopreserve sperm from the endangered Louisiana pinesnake (Pituophis ruthveni). The mixture contained Lake’s buffer with 10% N,N-dimethyl formamide (DMF), 2% methanol, 5% clarified egg yolk, (v/v% final concentration) and was tested against 16 experimental mixtures containing variable concentrations and mixtures of diluents, extenders, CPAs, and additives. In addition, we investigated the effects of post-thaw incubation on sperm motility in TL HEPES supplemented with 10% fetal bovine serum (H10) alone or supplemented with caffeine. We found that the majority of our test additives did not significantly improve the post-thaw motility or viability of sperm. The best performing experimental CPA mixture contained Lake’s buffer with 10% DMF, 2% methanol, and 5% clarified egg yolk with the addition of 5 mg/mL bovine serum albumin (BSA), and post-thaw incubation in both H10 and H10 with caffeine showed improved forward motility. Cryopreservation of sperm from the Louisiana pinesnake improved with the addition of BSA to our base CPA mixture, and post-thaw incubation in H10 improved with caffeine.Cryopreservation of sperm to preserve the genetic diversity of declining populations is a promising technique to aid in the recovery of endangered species such as the Louisiana pinesnake (Pituophis ruthveni). However, this technique has been performed on only a handful of snake species and with limited success. Here, we tested a cryoprotective agent (CPA) mixture containing Lake’s buffer with 10% N,N-dimethyl formamide (DMF), 2% methanol, 5% clarified egg yolk, (v/v% final concentration) against 16 other CPA-treatment mixtures. These contained either Lake’s buffer or TEST egg yolk buffer as the base diluent with a penetrating or non-penetrating CPA on the post-thaw recovery of sperm motility and viability. We also investigated the effect of post-thaw incubation treatment in TL HEPES supplemented with 10% fetal bovine serum (H10) alone or with caffeine on post-thaw motility parameters. Sperm from 16 Louisiana pinesnakes was cryopreserved, and the effectiveness of the CPA treatment mixtures and post-thaw treatments was determined based on measurements of sperm motility and viability. Sperm cryopreservation significantly reduced initial post-thaw sperm quality for all of the extender treatments. Viability of sperm was best maintained when cryopreserved in an CPA treatment mixture containing Lake’s buffer with 10% DMF, 2% methanol, and 5% clarified egg yolk with the addition of 5 mg/mL bovine serum albumin (BSA). For several extender mixtures a similar percent of post-thaw motility was observed, but no forward motility returned in any post-thaw samples prior to incubation in dilution treatments. Following incubation in both post-thaw treatments, the percent of forward motility and the index of forward progressive movement improved significantly. Post-thaw dilution with H10 containing caffeine improved motility parameters over H10 alone, suggesting further investigation of post-thaw treatment in caffeine could be beneficial. Although, cryopreservation of sperm from the Louisiana pinesnake continues to present a challenge, post-thaw dilution and the addition of BSA to CPA mixtures provides areas for improving cryopreservation methods for this endangered species.

Vapour fast freezing with low semen volumes can highly improve motility and viability or DNA quality of cryopreserved human spermatozoa.

ObjectivesTo challenge a vapour fast freezing (VFF) cryopreservation procedure (conventional VFF) with several vitrification protocols and VFF conducted with small semen volumes (10 μl, microVFF), in order to implement a procedure for sperm banking in subjects with small sperm number.Materials and methodsConventional VFF was conducted with test yolk buffer (TYB) as freezing medium and 500 μl straws as carriers. MicroVFF was conducted with TYB and using tips or cell sleepers as carriers. Vitrification was performed with TYB or SpermFreeze as freezing medium and with microspheres and tips as carriers. The effect of different procedures on progressive and total motility, viability, oxidative stress and DNA fragmentation of spermatozoa (sDF) was determined. Fresh and thawed samples, the latter after adequate washing/centrifuging, were evaluated. In some experiments, motility and viability recovery was determined in thawed samples, omitting the washing/centrifuging step.ResultsAll the cryopreservation procedures blunted sperm motility and viability and induced increase of oxidative stress and sDF. However, VFF better preserved sperm motility and viability and less induced oxidative stress and sDF than vitrification, independently from the freezing medium and the carriers used in the latter. MicroVFF with cell sleepers resulted in a percentage increase of 57.58 ± 63.63%, 48.82 ± 74.96% and 24.55 ± 39.20% of, respectively, progressive and total motility and viability compared to the conventional VFF. Further, when tips were used, microVFF resulted in a percentage decrease of 15.77 ± 20.77% of sDF with respect to conventional VFF. Finally, omission of washing/centrifuging in post thawed samples, resulted in a much lower negative effect on motility and viability.Discussion and conclusionVFF, and in particular microVFF, better prevents sperm cryodamage than vitrification. Washing/centrifuging step after sample thawing seems to be responsible for a relevant fraction of damage to sperm motility and viability. Overall, our results are promising for developing a novel strategy of sperm banking in subjects with small sperm number, where low semen volumes are mandatory.

How can mating systems inform future biobanking strategies? An illustration using two Indonesian bovids, banteng (Bos javanicus) and lowland anoa (Bubalus depressicornis)

Storing cryopreserved spermatozoa in a genome resource bank safeguards against the loss of heterozygosity in endangered species and provides opportunities to reincorporate genes into populations through the application of assisted reproductive technologies. The aim of this study was to demonstrate the effect of breeding strategy on ejaculate characteristics to illustrate how this information may be used to select appropriate methods for the storage and use of cryopreserved sperm. In the present study, ejaculates from a polygynous bovid, banteng (Bos javanicus), were characterized (motility 72.7 ± 4.3%; total sperm count 2,702 ± 764 ×106 sperm; morphologically normal sperm 87.9 ± 3.0%), as well as ejaculates from a monogamous bovid, lowland anoa (Bubalus depressicornis; motility 47.5 ± 5.4%; total sperm count 279 ± 84 ×106 sperm; morphologically normal sperm 69.0 ± 6.1%). As banteng produce an ejaculate with characteristics similar to domestic cattle, translating assisted reproductive technologies from domestic cattle is feasible. By contrast, lowland anoa produce smaller quantities of sperm with a higher prevalence of morphologically abnormal sperm; thus, alternative protocols, optimized for the storage and use of ejaculates containing lower quantities of sperm, is necessary. Sperm tail length was more conserved in banteng (CV 2.7%) than lowland anoa (CV 6.4%) and could be due to differences in levels of sperm competition between species. Additionally, the use of three different diluents (Biladyl, TES-Tris yolk buffer, and whole milk) were investigated for banteng sperm cryopreservation. Sperm cryopreserved in Biladyl and whole milk diluents produced significantly higher post-thaw survival parameters then TES-Tris yolk buffer.

Evidence of Spermatogenesis in the Presence of Hypothalamic Suppression and Low Testosterone in an Adolescent Transgender Female: A Case Report.

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. 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 patient's consent was obtained for written publication. 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. 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.

P–098 Use of Dimethylxanthine Theophylline in surgical retrieved sperms that do not recover motility after thawing

Abstract Study question Can the use of Theophylline recover motility of frozen surgically retrieved sperms in case of absence of motility after thawing? Summary answer Theophylline allows to recover motility of thawed surgically retrieved sperms. The utilization of sperms with or without pharmacological activation gives comparable clinical outcomes. What is known already Testicular sperm motility is usually poor. A method is needed to detect viable sperm for ICSI when motility is totally absent after freezing/thawing. Hypo-osmotic swelling test, mechanical touch technique, laser-assisted immotile sperm selection, birefringence-polarization microscopy and exposure to pharmacological stimulation are techniques used for this purpose. Among pharmacological agents Dimethylxanthine Theophylline is a phosphodiesterase inibitor that improves sperm motility by promoting an increase in intracellular cyclic AMP levels. Few studies report that it is efficient for recovery of sperm motility in cases of thawed testicular and retrograde ejaculation samples improving reproductive outcomes. Study design, size, duration Retrospective analysis of sixty frozen surgical sperm cycles (45 patients) utilized from February 2018 to November 2020. After thawing, samples were divided in two Groups according to motility recovery. Group A: presence of motility, Group B: absence of motility. Group B was treated with Theophilline and motility was re-assessed after incubation. Activated sperms were utilized for ICSI when available. Sperm motility recovery, fertilization, pregnancy rate/transfer, implantation and miscarriage rate were evaluated in both Groups. Participants/materials, setting, methods Surgical specimens were treated and concentrated in SpermRinse™ Medium (Vitrolife) and then cryopreservated in nitrogen vapor in TEST Yolk Buffer (Irvine Scientific). After thawing, only samples with no motility recovery were treated with a brief incubation in Theophylline (GM501 SpermMobil, Gynemed) and washed in Polyvinylpyrrolidone (ICSITM Vitrolife) before injection. ICSI was performed in all cases approximately 4–5 hours after sperm thawing. After fertilization check, transfer was scheduled in day 2. Main results and the role of chance Women’s age Group A (34,39±2,29 M±SD) and group B (35,87±4,34 M±SD) and men’s age Group A (37,31±5,12 M±SD) and group B (40,89±8.15 M±SD) were not significantly different (P= .328 and P=.218) respectively. Group A: 13/60 cycles (21.7%) (9 patients). Pre freezing and post thawing total motility percentage were 34.0±19.0 (M±SD) and 13.5±15.6 (M±SD) respectively (39.8% recovery). Group B: 47/60 cycles (78.3%) (36 patients). Pre freezing total motility percentage was 5.3±8.5 (M±SD) and no motility was recovered post thawing (0%). After treatment with Theophylline total motility was 1.8±1.8 (M±SD) (33.5% recovery). Motile sperms were utilized in all cases except from two in the Group B. Number of injected oocytes was 2.8±1.1 (M±SD) in Group A and 4.3±3.1 (M±SD) in Group B (P=.004) respectively. Fertilisation rate (63.1% and 45.4%, P=.066), Number of embryos transferred (1.8±0.7 M±SD and 1.6±0.7 M±SD, P=.271), Pregnancy rate/Transfer (54.5% and 37.1%, P=.502), Implantation rate (30.0% and 27.8%, P=.919) and Miscarriage rate (33.3% and 30.7%, P=.675) were not statistically significant between Group A and B respectively. In the two cases of group B injected with immotile sperm, fertilization rate was 0% (0/3) and 50% (2/4). Limitations, reasons for caution A larger study is needed to investigate the recovery of sperms motility (and/or their activation) and clinical outcomes, in particular referring to the origin of sampling (epididymal aspirate and testicular tissue) and type of azoospermia (obstructive and non-obstructive). Wider implications of the findings: Theophylline is an effective tool for sperm motility recovery after thawing allowing to inject viable sperm and facilitating laboratory handling. Trial registration number Not applicable

Carboxylated Poly-l-Lysine as a Macromolecular Cryoprotective Agent Enables the Development of Defined and Xeno-Free Human Sperm Cryopreservation Reagents.

In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive medicine, human serum albumin is frequently used because of low risks of zoonoses and allergic reactions. However, the risk of allogeneic infectious diseases exists, and the supply may be unstable because human serum albumin is derived from human blood. Therefore, the development of xeno-free human sperm cryopreservative reagents that could overcome the aforementioned problems is warranted. We succeeded in developing a new xeno-free and defined sperm cryopreservation reagent containing glycerol, carboxylated poly-l-lysine, and raffinose. The cryopreservation reagent was not significantly different in terms of sperm motility, viability, and DNA fragmentation and was comparable in performance to a commercial cryopreservation reagent containing human serum albumin. Moreover, the addition of saccharides was essential for its long-term storage. These results may help elucidate the unknown function of macromolecular-weight permeating cryoprotective agents.

Sperm cryopreservation in the Burmese python Python bivittatus as a model for endangered snakes.

Burmese pythons Python bivittatus captured in the Florida Everglades as part of an invasive species monitoring program served as a model for the development of sperm cryopreservation protocols for endangered snakes. Spermatozoa were collected from the vas deferens and initial motility, plasma membrane integrity and acrosome integrity were recorded before cryopreservation. Spermatozoa were extended in TES and Tris (TEST) yolk buffer with glycerol (GLY) or dimethyl sulfoxide (DMSO) concentrations of 8%, 12% or 16%, or combinations of GLY and DMSO with final concentrations of 4%:4%, 6%:6% or 8%:8%, and frozen at a rate of 0.3°C min-1 . Sperm frozen in combinations of GLY and DMSO exhibited greater post-thaw motility and plasma membrane integrity than those frozen in GLY or DMSO alone. All DMSO and GLY:DMSO treatments preserved a greater proportion of intact acrosomes than GLY alone. To determine the best overall cryopreservation protocol for this species, a sperm quality index was calculated, giving equal weight to each of the three measured indicators of cryosurvival. This analysis revealed that Burmese python spermatozoa frozen in 6% GLY:6% DMSO or 4% GLY:4% DMSO exhibited the highest post-thaw viability. This study represents the first comparative, comprehensive attempt to develop a sperm cryopreservation protocol for any snake species.

Sperm cryopreservation in an Australian skink (Eulamprus quoyii).

Assisted reproductive technologies for population and genetic management for threatened herpetofauna have grown substantially in the past decade. Here we describe experiments to optimise sperm cryopreservation in a model squamate, the eastern water skink Eulamprus quoyii . Small, concentrated volumes of highly motile spermatozoa were reliably collected from adult male E. quoyii by non-lethal ventral massage. Samples were used to: (1) test whether protein-rich diluents, namely Beltsville poultry semen extender (BPSE) and TES and Tris (TEST) yolk buffer (TYB), improve post-thaw quality metrics compared with Dulbecco's phosphate-buffered saline (DPBS); and (2) compare the efficacy of these diluents in combination with either 1.35M glycerol or 1.35M dimethyl sulfoxide (DMSO) at two freezing rates, fast (approximately -20°C min-1 ) versus slow (-6°C min-1 ). Glycerol and DMSO performed equally well in preserving spermatozoa under slow freezing rates. Under these conditions, the use of the complex diluents BPSE and TYB significantly improved post-thaw total motility compared with DPBS. Complex interactions occurred between cryodiluent type, cryoprotectant and freezing rate when testing fast versus slow freezing rates among treatment groups. Under slow freezing rates, DMSO was better at preserving membrane integrity and motility, regardless of diluent type, but successful fast freezing required complex diluents to support motility and membrane integrity, which has implications for implementation in a field setting.

Challenges in the development of sperm cryopreservation protocols for snakes.

Snake populations are declining worldwide, but research devoted to the development of sperm cryopreservation techniques for this taxon is very limited. Spermatozoa were collected postmortem from snakes of four squamate families (Elapidae, Colubridae, Viperidae and Pythonidae). Viability assessment was performed before and after cryopreservation. Spermatozoa were extended in TES and Tris (TEST) yolk buffer with 12% dimethylsulfoxide (DMSO) or 12% glycerol and frozen at a rate of 0.3°Cmin-1 . The sperm quality index (SQI), representing three viability parameters (motility, plasma membrane and acrosome integrity), was determined. Despite some species differences, glycerol was a more effective cryoprotectant for Colubridae, whereas DMSO provided greater cryoprotection for spermatozoa of members of the other three families.

Melatonin improves cryopreservation of ram sperm by inhibiting mitochondrial permeability transition pore opening.

Cryopreservation damages permeability of sperm mitochondrial membranes, with formation of a mitochondrial permeability transition pore (mPTP). Mitochondria are both a primary synthesis site and principle target for melatonin, which can directly inhibit mPTP formation. The objective was to determine effects of melatonin on mPTP opening of frozen-thawed ram sperm and elucidate underlying pathways by antagonist and agonists of melatonin receptors (MTs), and antagonists of PI3K and GSK 3β treatments; furthermore, plasma membrane integrity, mitochondrial membrane potential (ΔΨm), mitochondrial cytochrome c (Cytc) release and fertilization were analysed to assess the effect of mPTP status mediated by melatonin on quality of frozen-thawed sperm. Fresh ram semen was diluted in glucose-egg yolk buffer with 0 or 10-7 M melatonin (frozen and frozen+melatonin groups, respectively) and slow-frozen. In frozen-thawed sperm, melatonin added at initiation of 4°C equilibration was most effective for inhibiting mPTP opening, decreasing peptidyl-prolyl-cis/trans isomerase activity of cyclophilin D and increasing plasma membrane integrity, ΔΨm, mitochondrial Cyt c concentration and fertilizing ability (p<.05). In a mechanistic study, the melatonin receptor (MT)1 antagonist eliminated inhibition of melatonin on mPTP opening, whereas MT1 agonist had opposite effects (p<.05). Neither MT2 antagonist nor agonist had significant effect, but PI3K and/or GSK 3β antagonist decreased inhibition of MT1 agonist on mPTP opening (p<.05). In conclusion, melatonin improved sperm cryopreservation, perhaps by acting on MT1 via the PI3K-Akt-GSK 3β pathway to inhibit mPTP opening.

Melatonin Inhibits Formation of Mitochondrial Permeability Transition Pores and Improves Oxidative Phosphorylation of Frozen-Thawed Ram Sperm.

Structural and functional damages to mitochondria of frozen-thawed sperm are a typical cryoinjury, with mitochondrial permeability transition pore (MPTP) formation being the hallmark change. Mitochondria are both a primary synthesis site and principle target for melatonin; this compound can directly inhibit MPTP formation and therefore confer protection at a mitochondrial level. The objective was to determine effects of melatonin on MPTP opening, viability, motility, and oxidative phosphorylation (OXPHOS) of frozen-thawed ram sperm. Ram semen was diluted in glucose-egg yolk buffer with 0 or 10−7 M melatonin (frozen and frozen + melatonin groups, respectively) and slow frozen, with fresh semen as Control. In frozen-thawed sperm, melatonin inhibited MPTP opening and lactate concentrations and improved sperm viability, motility, acetyl-CoA concentration and adenosine triphosphate (ATP) production. With regard to the underlying physiological mechanism, melatonin suppressed movement of citrate synthase, isocitrate dehydrogenase, oxoglutarate dehydrogenase complex, and F0F1-ATP synthase permeability from mitochondrial to cytosolic fractions induced by MPTP opening; furthermore, it increased mRNA expressions of respiratory chain complex components and activities of complexes I, II, III, and IV and thereby improved oxygen consumption capacity in frozen-thawed sperm. In conclusion, melatonin improved OXPHOS of frozen-thawed ram sperm, attributed to inhibition of cryopreservation-induced MPTP opening.

229 From head to tail: A red wolf sperm project

Development of assisted reproductive technologies for the critically endangered red wolf (Canis rufus) is crucial to the maintenance of genetic diversity to support species recovery. Towards this goal, a cryopreservation protocol has previously been developed for red wolf sperm; however, the ability of the gametes to undergo capacitation has not been assessed in this species. Previously, we have shown that oviductal extracellular vesicles (oEVs) improve cat sperm motility and fertilizing ability. The objectives were to (1) compare the effects of culture media on motility and acrosomal integrity of fresh sperm, and select the best medium that can be used in a capacitation protocol; (2) identify potential biomarkers for sperm cryo-tolerance; and (3) determine the influence of canine oEVs on sperm survival and motility post-thaw. In Study 1, sperm were collected by electro-ejaculation from adult red wolves (n=8) and immediately cryopreserved in TRIS-egg yolk buffer with 8% glycerol or incubated for 18h in 5% CO2 and 38.5°C in one of the following media: canine capacitation medium (CCM), FERT-TALP (FERT), NCSU, synthetic oviductal fluid (SOF) and TRIS. At 0, 1, 2, 3, 4, and 18h, sperm were evaluated for total motility and acrosome integrity (FITC-PNA). In Study 2, sperm with high (&amp;gt;80%, HM, n=2 wolves) and low (&amp;lt;15%, LM, n=2 wolves) motility post-incubation at 4°C in the cryopreservation medium for 18h were subjected to proteomic analysis. In Study 3, oviducts were collected from domestic dogs (1-9 years, n=12) after elective spaying, and oEVs from various stages of the oestrous cycle [early follicular (EF), late follicular (LF), early luteal (EL), and late luteal (LL)] were isolated using the Total Exosome Isolation kit (Invitrogen). Frozen-thawed red wolf sperm (n=4 males) were incubated with 30×106 oEVs in non-capacitating CCM, and assessed as in study 1 at 0, 0.5, 1, 2, 3, 4, 6, 8, and 10h. Data were analysed using a paired samples t-test with 95% CI (Prism8, GraphPad Inc.). Sperm incubated in CCM and NCSU had higher motility than those in FERT, SOF, and TRIS after 2h of incubation and onward (2 h: 65±6, 68±6, 42±10, 57±8, and 43±5; 3 h: 60±9, 63±8, 36±11, 46±9, and 34±6; 4 h: 60±9, 60±10, 30±10, 43±8, and 20±5; 18 h: 12±7, 15±7, 9±5, 3±2, and 0, respectively; P&amp;lt;0.05). After 1h of incubation, samples incubated in CCM, NCSU, and SOF had a higher number of sperm with intact acrosomal membranes (P&amp;gt;0.05) than other treatments. A total of 179 proteins were identified, of which 129, including those regulating energy metabolism and mitochondrial mediated apoptosis, were differentially expressed between HM and LM. Preliminary data from Study 3 suggested that thawing and incubating sperm in the presence of LF, EL, and LL oEVs improved sperm motility. In conclusion, CCM and NCSU sustained sperm survival after invitro incubation and could be candidates for invitro fertilization studies in the red wolf. Data generated from sperm proteomic analysis provided insights into cellular pathways regulating sperm cryo-sensitivity. Finally, we demonstrated the potential of oEVs in improving wolf sperm survival post-thawing.

Semen cryopreservation in golden-headed lion tamarin, Leontopithecus chrysomelas.

Wild animal genetic resource banking (GRB) represents a valuable tool in conservation breeding programs, particularly in cases involving endangered species such as the golden-headed lion tamarin (Leontopithecus chrysomelas). Thus, we aimed to assess a sperm freezing protocol for golden-headed lion tamarins using two different exenders: BotuBOV® (BB) and Test Yolk Buffer® (TYB). Ejaculates were collected by penile vibrostimulation from animals housed at São Paulo Zoological Park Foundation, São Paulo, Brazil, and after immediate analysis, two aliquots were diluted in BB and TYB. Postthawing samples were evaluated for total and progressive motility, plasma membrane and acrosome integrities, mitochondrial activity, susceptibility to oxidative stress, and sperm-egg-binding. No differences between BB and TYB were found for most seminal parameters, except for acrosome integrity and susceptibility to oxidative stress (in both cases BB showed higher values). However, in spite of these differences and regardless of the extender used, postthaw sperm motility and viability with the described protocol were encouraging (on average >50% and >80%, respectively), indicating that sperm cryopreservation may be a short-term measure for the conservation of golden-headed lion tamarins.

Cryopreservation of margay (Leopardus wiedii) spermatozoa: Effects of different extenders and frozen protocols

Sperm cryopreservation offers many benefits to wild felids conservation programs. However, the implementation of these programs is limited by the different responses of each species to the cryopreservation protocols and extenders used, requiring the formulation of species-specific protocols. For this purpose, semen samples from 6 margays (Leopardus wiedii) were submitted to 2 cryopreservation protocols: 1) manual freezing (cooling rate of – 0.33 °C/min at 5 °C/180 min and freezing rate with two steps – 9 °C/min for 2 min and −19.1 °C/min for 2 min) and 2) automatic freezing machine (cooling rate of – 0.25 °C/min at 5 °C/120 min and freezing rate with one step −20 °C/min for 8.3 min) using 2 commercial extenders, an egg yolk-based (Test Yolk Buffer; TYB) and an egg yolk-free extender (AndroMed; MED). Post-thawed sperm quality was assessed at 3 time points (immediately after thawing and 1 and 2 h post-thawed) by sperm motility index (SMI), plasma membrane and acrosomal integrity, and mitochondrial membrane potential (MMP). Regarding SMI, TYB yielded superior results (29.4 ± 3.5%) compared to MED (11.2 ± 2.8%; p < 0.002) immediately after thawing until 2 h after thawing (TYB 3.9 ± 1.7% and MED 0.0 ± 0.0%; p < 0.05). Furthermore, the automated freezing method provided higher motility compared to the manual freezing procedure immediately post-thaw (25.08 ± 3.66% and 15.78 ± 3.29%, respectively) and 1 h post-thaw (13.71 ± 2.56% and 6.03 ± 1.97%, respectively; p < 0.05). The percentage of intact acrosomes and plasma membranes and the percentage of sperm with high MMP were superior for TYB when compared to MED regardless of cryopreservation protocol (p < 0.05). Conversely, the interaction between cryopreservation protocols and extenders was observed for MMP where TYB exhibits better results compared to MED (p < 0.05) in both procedures, but it was higher in automated procedures. For MED, no changes were found in MMP between procedures. Considering only TYB, samples showed higher MMP when submitted to an automated procedure (p < 0.05). In conclusion, the slow cooling rates with shorter time of exposure to glycerol contributed to minimize cryodamage in the Margays’ sperm. Moreover, results indicated that association between TYB and automatic freezing machine ensured the minimal quality of spermatozoa after thawing required for further use in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).

Assessment and preservation of liquid and frozen-thawed Black crested mangabey (Lophocebus aterrimus) spermatozoa obtained by transrectal ultrasonic-guided massage of the accessory sex glands and electroejaculation

The Black Crested Mangabey (Lophocebus aterrimus) is an African monkey listed as Near Threatened by the IUCN and in captivity the population is limited to 34 males. The aim of this study was to evaluate two Black Crested Mangabey males, maintained in captivity in a zoological garden and suspected of infertility, with a complete examination of their genital tract using ultrasonography, followed by recovery of semen using transrectal ultrasonic massage of the accessory sexual glands (TUMASG) and electroejaculation. One male had small testicular and accessory sex gland sizes indicative of senile hypoplasia. The other male was suspected of infertility. Four semen samples were obtained. Fresh semen was initially evaluated, diluted in Refrigeration Medium Test Yolk buffer, cooled at 15 °C and cryopreserved. Endocrine profiles (testosterone, oestradiol, FSH, LH, cortisol), prostatic specific antigen and semen variables (volume, concentration, motility by CASA, viability and acrosome status using flow cytometry, morphology, morphometry utilising TEM) were evaluated in raw, cooled and cryopreserved samples. There was no detrimental effect of cooling or cryopreservation on sperm viability and acrosomal integrity. Similar percentages of viable and acrosome-intact spermatozoa were present in cooled (for 6 h) and frozen-thawed semen samples (75.1% compared with 69.0%, P > 0.05), while progressive motility was greater in cooled, compared with frozen-thawed samples (81.5% compared with 67.3%). This study was the first in which there was evaluation of sperm variables in this species and, although this study is limited by the number of animals it provides background information for further studies using assisted reproductive technologies.

Impacts of test (TES and Tris) yolk buffer and cooling on the ability of human sperm to capacitate

Impacts of test (TES and Tris) yolk buffer and cooling on the ability of human sperm to capacitate

Prolonged semen cryopreservation decreases motile concentration

There is a paucity of data regarding the viability of cryopreserved sperm. Good laboratory practice involving cryopreservation include QA/QC, equipment maintenance and stable temperatures of -196°C. These ensure tissues remain viable for later use. However, cryopreservation has been shown to alter the structure and function of stored spermatozoa. This study aims to determine if sperm viability is affected by long-term storage. Before-After Study. Patients sperm samples that were abandoned over the years of 1995-2014 (average 14.5 years) were thawed and analyzed before discard. Samples were considered abandoned if patients couldn’t be contacted within the past 5 years to continue storage. Prior to initial freeze, semen analyses were performed. Semen samples were stored in a 1:1 mixture of test yolk buffer, aliquoted into a 1.5 ml cryovial and suspended in nitrogen vapor for 30 minutes prior to being plunged into liquid nitrogen. Vials were thawed by placing cryovials into 37°C heat blocks for 20 minutes. Post-thaw survival of sperm was determined by calculating sperm concentration, motility and progression. Pre and post analyses were analyzed using regression analyses with a cluster analysis to account for pairwise comparisons. 131 patient semen samples were grouped into four categories listed in Table 1. Age, initial sperm concentration and motility were not different between groups. Overall there is a significant decline in sperm motility with years of storage as vials stored for 20 years and longer show a 70% decrease compared to 10 years and less (p=0.0012). The patient age or initial sperm concentration at the time of freeze has no impact on sperm survival. Despite appropriate measures to maintain specimen in storage, it appears that prolonged storage in liquid nitrogen may impact sperm survival. This result warrants further study as viability of tissues may be affected over time and reduce the success of fertility outcomes.Table 1Summary of difference between initial and post-thaw motile concentrationsYears StorednPt AgeInitial Motile Concentration (M/ml)Post-Thaw Motile Concentration (M/ml)Δ<103038.8±4.937.6±33.519.6±29.80.26±0.2610-143637.7±7.528.2±29.316.6±220.28±0.1815-193638±7.768.8±3610.1±21.90.18±0.21>202934.7±8.745±58.211.7±24.10.09±0.13**p<0.05 Open table in a new tab