Post-thaw Total Motility Research Articles

Seminal oxidative stress index can be used as a marker in the prediction of bull semen cryotolerance

In this study, it was aimed to evaluate the effect of seminal plasma activity levels of enzymatic antioxidants such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), non-enzymatic antioxidant alpha-tocopherol (Vit-E), and also total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) on post-thaw sperm quality. As well as it was aimed to investigate the possibility of the use of OSI as a marker for the estimation of bull semen freezability. For this study, 72 ejaculates were collected from 6 bulls and separated into two aliquots. The first one was centrifuged to separate seminal plasma. The latter one was cryopreserved and stored in liquid nitrogen until analysis. Post-thaw semen quality was examined in two groups (good-freezable semen (GFS) and poor-freezable semen (PFS)) through cluster analyses based on post-thaw total motility and plasma membrane and acrosome integrity. As a result of the analyses, seminal TAS, CAT, and Vit-E values were higher (P<0.05) in the GFS group, while TOS and OSI values were higher (P<0.01) in the PFS group. We also performed an ROC curve analysis to determine whether the seminal OSI value could be used to predict semen freezeability. The area under curve (AUC) value was found as 0,70 (P=0.006). In conclusion, it has been revealed that the seminal plasma antioxidant content is responsible for the freezability of semen, and the OSI value, which can be determined by performing TAS and TOS analyses instead of looking for separate antioxidant enzymes, can be used as a marker for the estimation of post-thaw semen quality at artificial insemination centers.

First dose optimization study on freezing Anatolian buffalo semen

The main objective of sperm production centers is to produce as many straws as possible from the obtained ejaculates using the optimal dilution rate. To this end, this study is the first to evaluate the effect of different semen extender rates on Anatolian buffalo semen quality. Ejaculates were collected by artificial vagina from three Anatolian buffalo bulls. These ejaculates were divided into three aliquots and filled into 0.25 ml straws with soy-based extenders at concentrations of 35, 25, and 15 million sperm/straw (n=105). The straw samples of different sperm concentrations were frozen. The quality of sperm was evaluated after thawing (37 °C, 30 sec) and following the thermoresistance test (37 °C, 3 h). The post-thaw total motility and progressive motility values were similar between the groups. However, following the thermoresistance test, there was a significant decrease in total motility in the 35 million sperm/straw group, and the progressive motility was significantly higher in the 25 million sperm/straw group. There was no statistically significant difference between the groups in terms of sperm kinetic parameters, except for VSL after thawing, as well as VAP and LIN values following the thermoresistance test. The overall mean PMAI and STR values were the highest in the 25 million sperm/straw group. In conclusion, it is recommended to dilute the Anatolian buffalo semen at a concentration of 25 million/0.25 ml when freezing it with a soy-based semen extender. In addition, it is considered that soy-based extenders compensate for cryo-damage to sperm motility for a short time, and the thermoresistance test should be applied for objective evaluation in dose optimization studies.

Factors affecting the analysis and interpretation of sperm quality in frozen/thawed stallion semen

In the current study, we examined: 1) the agreement (bias) between fluorescence-based methods (NucleoCounter-SP100 [NC] vs. flow cytometry [FC]) for determining the viability (VIAB) of frozen/thawed stallion sperm; 2) the agreement between post-thaw sperm total motility (TMOT) and VIAB; 3) whether a difference between TMOT and VIAB [VIAB – TMOT] in frozen/thawed stallion sperm could be explained by the level of lipid peroxidation in viable sperm (VLPP); 4) the repeatability of post-thaw analysis of sperm quality; and 5) the effect of final post-thaw semen dilution (10, 30, or 50 x 106 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.05), and the agreement between these two methods was high (bias: 1 to −3). The agreement between post-thaw TMOT and VIAB decreased as the pre-freeze percentages of morphologically normal sperm and DNA quality decreased: bias – 4 to – 25. The bias between [VIAB – TMOT] and VLPP ranged from – 5 to 7. Differences in post-thaw sperm quality (TMOT, PMOT, VIAB, and sperm concentration) were not observed when analyzing one or three straws per ejaculate (P > 0.05). There was no effect of post-thaw sperm concentration (i.e., 10 vs. 30 vs. 50 x 106 sperm/mL) on sperm motion characteristics (P > 0.05). This study reports factors other than post-thaw sperm motility that warrant further consideration when analyzing frozen/thawed stallion sperm.

Ameliorative effect of crocin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Buffalo bull sperm suffer more cryoinjuries due to lipid peroxidation of high structural polyunsaturated fatty acid contents than cattle sperm. Consequently, the post-thaw fertilization potential of buffalo bull sperm is compromised. Crocin is a carotenoid known for its antioxidant potential through scavenging reactive oxygen species. Objectives of the current study were to investigate the effect of crocin addition in the semen extender on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm. Semen samples (n = 32) from four Nili-Ravi buffalo bulls were extended with tris-citric acid extender containing different concentrations of crocin (0 mM; control, 0.5, 1, 1.5 and 2 mM). The extended semen was packed in 0.5 mL French straws (25 × 106 sperm/straw) and cryopreserved in liquid nitrogen. Computer-assisted semen analysis, hypo-osmotic swelling test, normal apical ridge assay, Rhodamine 123, acridine orange, propidium iodide staining, and thiobarbituric acid reactive substances assay were performed to assess sperm motility parameters, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability, and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. The fertility of semen doses containing the most potent concentration of crocin (based on optimum post-thaw semen quality) was compared with control during the breeding season. Buffaloes (n = 400; 200/group) were inseminated 24 h after the onset of oestrus and transrectally palpated for pregnancy at least 60 days post-insemination. Results revealed that 0.5 and 1 mM crocin improved sperm post-thaw total motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential and viability, and 1 and 1.5 mM crocin enhanced catalase activity and reduced lipid peroxidation compared to control (p < .05). Moreover, 1 mM crocin improved sperm post-thaw progressive motility, kinematics, and DNA integrity, and 1.5 mM crocin enhanced plasma membrane integrity than control (p < .05). Expression levels of SPACA3, PRM1 and BCL2 genes were higher (p < .05) with 1 mM crocin compared to other groups. In contrast, no difference (p > .05) was noticed in expressions of BAX and ROMO1 genes among all groups. The fertility rate of semen doses containing the most potent concentration (1 mM) of crocin was higher (p = .0465) compared to control (56 ± 0.03% vs. 46 ± 0.04%, respectively). In conclusion, 1 mM crocin in the semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm.

Phosphatidylcholine and cholesterol-loaded-cyclodextrins can replace egg yolk in the cryopreservation of equine spermatozoa

In the horse breeding industry, cryopreservation of spermatozoa has become an integral aspect of modern-day protocols. To avoid sperm damage during the freezing process, freezing media containing egg yolk are used. Recently, urgency to find an alternative to egg yolk has been increased due to the prevalence of Avian Influenza Virus (AIV). In the UK, the World Organization for Animal Health reports that between July and December 2022, an AIV outbreak led to the death of 4.62 million birds (56.73 million globally), with substantial consequences for agriculture, environment and egg availability (WAHIS. 2023; https://wahis.woah.org/#/dashboard/qd-dashboard: Accessed: 12 January 2023). Recent research has also called into question the biosafety of egg yolk, with RNA from highly pathogenic and low pathogenic AIV found on and in eggs (Stephens.et.al. Avian Diseases. 2020; 64:143-148). The cryoprotectant potential of phosphatidylcholine, the main phospholipid in egg yolk, has been investigated in diluents used for cryopreservation of human spermatozoa with some success (Sicchieri.et.al. Translational Andrology and Urology. 2021; 10:397-407). The chemically defined origin and increased biosafety make phosphatidylcholine a desirable alternative to egg yolk for use in cryopreservation. Ejaculates from four horses (n=4) were frozen using four different freezing extenders: a reference medium containing egg yolk and 3% dimethylformamide (control) and media without egg yolk supplemented with 1.5mg/ml2-hydroxypropyl-β-cyclodextrin cholesterol complex, 3% dimethylformamide and either 2% (P1), 4% (P2) or 6% phosphatidylcholine (P3). Samples were diluted and centrifuged on a density gradient medium (1000xg’ for 20 minutes). The cell band was aspirated and resuspended (200 × 10⁶ cells/ml) with allotted treatment extender. Samples were packed into 0.25ml straws, cooled to 5˚C for 25 minutes, then frozen using a programmable freezing unit (IceCube14s, Minitube, Germany). Temperature of the straws was reduced from 4 ˚C to -143˚C in 9 minutes, after which they were plunged directly into liquid nitrogen (-196˚C). Viability was assessed using NucleoCounter® NC-100™. Total motility analyses were performed with computer assisted semen analysis (Hamilton Thorne, USA). No difference in post-thaw viability was found between any of the media tested (P>0.05). No difference in total motility was found between the control (36.75±1.93σM), P1 (29.25±3.82σM) or P2 media (19.5±6.35σM) (P>0.05). However, post-thaw total motility of sperm cryopreserved in P3 media (16±6.01σM) was lower than the control (P<0.05). Media supplemented with 2-4% phosphatidylcholine and 1.5mg/ml cholesterol was effective in limiting loss of total motility and membrane integrity of equine spermatozoa, establishing the need for further breeding trials into the use of phospholipids for egg yolk lipid replacement to improve biosafety. The authors wish to recognize the support of Tullis Matson and Stallion AI Services Ltd in this study.

An Investigation of Equine Sperm Quality Following Cryopreservation at Low Sperm Concentration and Repeated Freeze-Thawing

Stallion spermatozoa are typically cryopreserved at 200 to 300 million sperm/ml; however recent advances such as intracytoplasmic sperm injection (ICSI) requires only one spermatozoon, wasting many, after thawing a whole straw. Cryopreserving at concentrations less than the current standard or refreezing thawed spermatozoa could maximize the use of genetically valuable animals and reduce waste. This investigation aimed to identify if lowering the sperm concentration for cryopreservation affected post-thaw quality after one and two freeze-thaw cycles. Nine ejaculates were collected from three fertile, “good freezer” stallions (post-thaw motility ≥35%) for experiment 1. Each ejaculate was split into eight treatments: five, 10, 20, 50, 100, 200, 300, 400 million sperm/ml and cryopreserved. Post-thaw: motility, viability, acrosome integrity and oxidative stress were assessed. Experiment 2, straws from experiment 1 (300 million sperm/ml) were thawed, diluted to 20 million sperm/ml or left undiluted (control) and refrozen. Post-thaw motility and viability were assessed. In experiment 1 sperm concentration did not affect post-thaw total motility (TM), progressive motility (PM) or viability at 50 to 400 million sperm/ml (P > .05). Whilst sperm concentrations of five to 20 million/ml did differ (post-thaw TM and PM). Both refreezing and reducing spermatozoa concentration, decreased TM, PM and viability (P < .05) after two freeze-thaw cycles. These results suggest cryopreserving at sperm concentrations as low as 50 million/ml maintains spermatozoa quality in good freezer stallions. Spermatozoa maintained some motility and viability when initially cryopreserved at 20 million sperm/ml and after two freeze-thaw cycles but research should investigate more optimal conditions.

Retrograde Flushing Followed by Slicing Float-Up as an Approach to Optimize Epididymal Sperm Recovery for the Purpose of Cryopreservation in Equids.

Simple SummaryHarvesting epididymal sperm often represents the last opportunity to cryopreserve the breeding potential of a sire. Epididymal sperm harvesting is performed via retrograde flushing (RF) or slicing float-up (SF) of sperm into an extender. However, these techniques are not applied in combination unless RF results in poor yield. This study aimed to assess the yield and cryopreservation of epididymal sperm harvested via RF followed by SF in equids. The total sperm harvested by RF in donkeys resulted in the greatest yield, and horse epididymal harvesting via SF resulted in the fewest yield. The use of RF followed by SF resulted in 57% and 31% more sperm per harvest in the donkey and horse. Technique did not affect pre- and post-freezing sperm parameters. These results suggest that this could become a new standard approach in clinical practice.This study aimed to assess the parameters of epididymal sperm harvested by retrograde flushing (RF) followed by slicing float-up (SF). Epididymides from donkeys (n = 18) and horses (n = 28) were subjected to RF with a freezing extender and then SF technique. The retrieved sperm after RF and SF was evaluated for volume, concentration, and total sperm and then cryopreserved separately. Post-thaw total motility (TM) and progressive motility (PM) were evaluated with CASA. Sperm membrane integrity (SMI) and mitochondrial membrane potential (MMP) were assessed with flow cytometry. Sperm concentration was greater in donkeys than horses (684 ± 62.9 vs. 494 ± 50.9 million sperm/mL) (p = 0.02). The total sperm harvested was lower in SF (3.6 ± 0.7 billion) than RF (10.4 ± 1.5 billion) and in horses (4.6 ± 0.8 billion) than in donkeys (10.7 ± 1.8 billion) (p < 0.05). RF followed by SF resulted in 57% and 31% more sperm per harvest in donkeys and horses. Results of TM and PM before freezing were not affected by technique or species (p > 0.05). Post-thawing SMI and MMP did not vary with technique or species (p > 0.05); TM and PM were not influenced by the technique or the species (p > 0.05) but by their interaction (p = 0.005). In conclusion, using RF followed by SF enhances sperm recovery without affecting cryopreservation in equids.

Seminal Plasma Antioxidants Are Related to Sperm Cryotolerance in the Horse.

The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to harvest the SP and assess the activity levels of antioxidants and the OSI, and the second one was cryopreserved. The following post-thaw sperm quality parameters were evaluated: sperm motility, plasma membrane and acrosome integrity, mitochondrial membrane potential, intracellular levels of reactive oxygen species (ROS), and plasma membrane lipid disorder. Based on post-thaw total motility (TM) and plasma membrane integrity (SYBR14+/PI−), ejaculates were hierarchically (p < 0.001) clustered into two groups of good (GFE) and poor (PFE) freezability. The SP activity levels of PON1, SOD, and TEAC were higher (p < 0.05) in GFE than in PFE, showing a positive relationship (p < 0.05) with some sperm motility parameters and with plasma membrane (PON1 and TEAC) and acrosome (SOD and TEAC) integrity. In contrast, OSI was higher (p < 0.05) in the SP of PFE than in that of GFE, and was negatively correlated (p < 0.05) to some sperm motility parameters and to plasma membrane and acrosome integrity, and positively (p < 0.05) to the percentage of viable sperm with high plasma membrane lipid disorder. In conclusion, enzymatic (PON1 and SOD) and non-enzymatic (TEAC) antioxidants of SP are related to horse sperm cryotolerance. In addition, our results suggest that PON1 could be one of the main antioxidant enzymes involved in the control of ROS in this species. Further investigation is needed to confirm the potential use of these SP-antioxidants and OSI to predict sperm cryotolerance in horses.

Effect of graphene oxide as cryoprotectant on post-thaw sperm functional and kinetic parameters of cross bred (HF X Sahiwal) and Murrah buffalo (Bubalus bubalis) bulls

Graphene oxide (GO) greatly suppresses the growth and recrystallization by curving the hexagonal shape of ice crystals. Study was conducted to evaluate effect of GO as cryoprotectant in semen extender for augmenting sperm viability in dairy (cattle and buffalo) animals. In experiment one, semen was extended with TRIS Egg Yolk Glycerol (TEYG) extender supplemented with different concentrations of GO: 0.0125, 0.25, 0.5, 0.1 and 0.2 mg ml−1. Freezing of semen samples was conducted at 30 °C min−1 from temperature drop from 4 °C to −15 °C and −15 °C to – 60 °C followed by 50 °C min−1 from – 60 °C to −140 °C, and the semen straws were plunged in liquid nitrogen. Second experiment evaluated the performance of TEYG extender supplemented with combinations of GO (G05 as 0.05 and G10 as 0.1 mg ml−1) and glycerol (T48 as 4.8 and T64 as 6.4%) in four groups as G05T48, G05T64, G10T48 and G10T64. Freezing rates of 30 °C min-1[Protocol (PRT) I], 40 °C min−1 (PRT II) and 50 °C min−1 (PRT III) in the critical temperature fall zone of −15 °C to −60 °C were evaluated for semen extender supplemented with glycerol 6.4% and GO 0.05 mg ml−1 in the third experiment. Cattle (n = 3) and buffalo (n = 3) bulls were chosen for the study taking six ejaculates per bull per treatment. Post-thaw sperm motility, membrane integrity, viability and abnormalities were observed by means of CASA, Hypo-osmotic swelling test (HOST), Eosin-Nigrosin stain and Rose Bengal stain procedures, respectively. Post-thaw total motility (TM), progressive motility (PM), VCL, VSL, VAP, HOST response and viability increased significantly in extender with GO concentrations of 0.1 and 0.05 mg ml−1 as compared to control. Per cent abnormalities were significantly (p < .05) lower in group with GO 0.025 and 0.0125 mg ml−1 as compared to control. Results from the second experiment showed higher post-thaw TM, PM, VCL, VAP, VSL, HOST response, viability increased significantly (p < .05) in G05T64 and G05T48 as compared to G10T64. Sperm abnormalities did not vary among the groups as compared to control for cattle spermatozoa. In the third experiment post-thaw TM, PM, VCL, VSL, VAP, HOS response and sperm viability increased significantly (p < .05) in PRT III as compared to PRT I for buffalo and cattle spermatozoa. Sperm abnormalities were significantly (p < .05) lower in PRT II and PRT III as compared to PRT I for buffalo, whereas, lower in PRT II as compared to PRTI for cattle spermatozoa. GO as cryoprotectant when added to semen extender at the rate of 0.05 and 0.1 mg ml−1, resulted in better plasma membrane function and viability. Glycerol concentration below 6.4% in buffalo semen extender reduced post-thaw quality of sperm even when GO was added to the extender. Higher freezing rate of 50 °C min−1 in the critical temperature fall zone of −15 to −60 °C perform better than the freezing rate of 30 °C min−1. It is concluded that TEYG extender having glycerol 6.4% and GO 0.05 mg ml−1 improved post-thaw semen quality of cattle and buffalo.

Semen collection, evaluation, and cryopreservation in the bonobo (Pan paniscus)

BackgroundCaptive breeding of bonobos (Pan paniscus) has proven to be successful, but maintaining genetic diversity remains a challenge. Cryopreservation of semen is an important potential tool to maintain genetic diversity by preserving current genetic material for future use, as well as facilitating the transport and exchange of genetic material. This study aimed to develop a protocol for semen collection and cryopreservation in the bonobo. Semen was collected from four healthy adult bonobos under general anesthesia during management translocation procedures. Semen collection utilizing urethral catheterization was not successful (n = 1), however, all males (n = 4) responded well to rectal probe electro-ejaculation. Immediately after collection, ejaculates were evaluated for color and admixtures, volume, motility, and concentration. Eosin-Nigrosin staining was prepared to evaluate morphology and viability. Ejaculates were split into two equal volumes and cryopreserved in two different extenders, using a one-step and a two-step approach. Ejaculates were gradually cooled to 4 °C in two hours, subsequently stored in liquid nitrogen vapor for twenty minutes (0.25 ml straws), and finally dropped into liquid nitrogen.ResultsPre-freeze evaluation showed thick, white samples with an average ejaculate volume of 450 µl (100-1000 µl), total motility of 59% (40–80%), viability of 69% (38–85%) and 58% (46–72%) normal spermatozoa. Mainly head (22%) and tail (19%) defects were detected on the Eosin-Nigrosin stain. Ejaculates were highly concentrated, nevertheless, due to the coagulum that caused high viscosity and non-homogenous fractions, only estimations of concentration could be made (1000 million/ml). After 24 h of storage, the post-thaw evaluation showed a loss of quality with an average post-thaw total motility of 15% (5–25%) using the one-step freezing medium, and 19% (5–30%) using the two-step medium. Average post-thaw viability was 15% (4–24%) and 21% (15–29%), respectively.ConclusionsThis report on ejaculates from bonobos obtained by rectal probe electro-ejaculation shows that semen parameters of this species are not completely similar to those of its sibling species, the chimpanzee. Further studies are necessary to develop an optimal protocol for the processing and cryopreservation of bonobo spermatozoa.

P–126 A comparison of the efficacy of sperm freezing using high security tubes versus high security straws

Abstract Study question Are there any advantages in using High security tubes rather than High Security straws for conventional slow sperm freezing? Summary answer Freezing sperm in High Security tubes (HST) improved post-thaw recovery rate and motility, and also reduced processing and handling compared to High Security straws (HSS). What is known already The use of High Security freezing consumables (HSFC) in an IVF setting is a safe and effective way of eliminating concerns related to viral cross-contamination during storage. The lower diameter of HSS does make them susceptible to warming during handling. The HSFC used in this study is the only CE marked products that are made of resin, leak-proof and shatter-proof in all cryogenic temperatures even in LN2. No previous studies have compared the use of HST with HSS for conventional human sperm freezing. This study sets out to investigate the performance of HST compared to HSS. Study design, size, duration The study was designed as a controlled split-sample study with blind post-thaw analysis. Following the routine WHO analysis of 20 semen samples, the remainder of each of the samples was evenly divided and cryopreserved by conventional slow freezing in each of the two different HSFC. The freeze was conducted simultaneously by the same practitioner, employing the same freezing protocol and cryoprotectant. The pre-freeze and post-thaw concentration, total and progressive sperm motility were recorded. Participants/materials, setting, methods At one IVF clinic, semen samples with sperm density ≥15million/ml, ≥40% motility, ≥1.5ml were included. Cryoprotectant (SpermFreeze, Fertipro) was added dropwise to unprepared semen and kept at room temperature for 10 minutes before loading into HSFC (0.5ml CBS™HSS; CBS™HST). HSFC were heat-sealed (SYMS; SYMSIII sealers) and placed in vapour for 30 minutes before plunging into LN2. Samples were thawed by immersion in a 37Cº water bath for 5 minutes and analysed using WHO methods. Main results and the role of chance Paired-t test was used to compare the percentage motility between the different HSFC. All analysis was considered statistically significant when p &amp;lt; 0.01. We demonstrated that the sperm recovery rate (Percentage total motility post-thaw/ Percentage total motility pre-freeze) in HST was 66.63 ± 14.94 (mean ± standard deviation) compared to 40.80 ± 14.69 in HSS. In the HSS, the percentage post-thaw total motility was 19.99 ± 7.21 and the percentage post-thaw progressive motility was 12.26 ± 2.59. In the HST, the percentage post-thaw total motility was 32.57 ± 8.33 and the percentage post-thaw progressive motility was 23.08 ± 5.53. The overall improvement when using HST against HSS was 12.53 ± 5.69, 10.44 ± 5.29 for the total motility and the progressive motility respectively. Comments were recorded regarding the handling and the condition of the HSS and HST for each freeze event. Neither device displayed any leakage of LN2 or any explosion during the warming. The freezing process was easier and faster using HST rather than HSS. It was also noted that the entire sample can be recovered from the HST, unlike the HSS. Limitations, reasons for caution The study looked at sperm recovery in terms of motility only. DNA damage was not considered as a parameter of sperm quality. Also, fertilization, pregnancy rates, live birth rates and the use of poorer quality sperm samples have not been investigated. Wider implications of the findings: For conventional sperm freezing, the use of HST resulted in improved sperm motility and progression post-thaw, when compared to HSS. This finding supports the use of HST to improve the post thaw quality of sperm, benefitting patients with own frozen samples, recipients of donor sperm and donor sperm banks. Trial registration number Not applicable.

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.

Effects of antioxidants on motility and DNA integrity in frozen-thawed sperm

Aims: This study aims to investigate whether the addition of the antioxidant complex including taurine, ascorbic acid, and glutathione into the cryopreservation medium affects the damage to sperm during the freezing process. Methods: Ejaculate samples of patients who applied for semen analysis to the Assisted Reproduction Unit of Private Adatip Hospital were used. Fresh samples were analyzed for standard semen quality parameters according to the World Health Organization guidelines. Samples within the normal range were evaluated for sperm DNA fragmentation using the Halosperm technique. Remaining ejaculates were washed with the gradient method before cryopreservation. Sperm samples of each patient were divided equally for freezing in a cryopreservation medium with or without the antioxidant supplementation. One month later, the samples were thawed. Post-thaw total motility and DNA fragmentation were determined for each sample. Results: Semen samples of 40 patients were analyzed. We observed decreased total motility (34.8 ±5.32 % vs. 65.5 ±6.42 %, P= 0.002) and increased sperm DNA fragmentation (52.3±5.42 % vs. 26.4±3.12 %, P= 0.002) in post-thaw semen samples following cryopreservation in comparison to fresh samples. The addition of antioxidants to the freezing medium did not have a statistically significant effect on sperm motility (38.3± 6.22 % vs. 34.8 ±5.32 %, P =0.07) and DNA damage (47.5±4.7 % vs. 52.3±5.42, P =0.08) when compared to control samples following the freezing process. Conclusions: We observed increased sperm DNA fragmentation and decreased total motility following cryopreservation. No significant improvement in sperm motility or DNA integrity was obtained after the addition of 5 µM of the antioxidants taurine, ascorbic acid, and glutathione to the freezing media.

High levels of oxidation-reduction potential in frozen-thawed human semen are significantly correlated with poor post-thaw sperm quality.

This study examined the relationship between oxidation-reduction potential (ORP) in frozen-thawed semen and the post-thaw sperm parameters. Levels of ORP were measured in 25 samples from men presenting for routine infertility work-up and were expressed as millivolt (mV)/106 sperm/ml. Frozen-thawed samples were examined for post-thaw total motility (TM%), progressive motility (PM%), total sperm count (TSC) and ORP. The cryo-survival rate (CSR) was calculated as post-thaw TM/pre-freeze TM×100. Data are provided as median and interquartile range (25th and 75th percentiles). The post-thaw TM% (10.0% [4.00%, 15.1%]), PM% (5.88% [2.97%, 9.33%]) and TSC (12.5 [10.0, 17.5]×106 sperm) were significantly lower than the pre-freeze TM% (45.9% [32.9%, 59.1%], PM% (31.5% [24.4%, 40.0%] and TSC (120 [90, 250] ×106 sperm) (p<.001). Post-thaw ORP (2.62 [2.52, 3.13]mV/106 sperm/ml) was significantly higher than pre-freeze ORP (0.73 [0.54, 1.21]mV/106 sperm/ml; p<.001). The CSR was 21.7% (11.3%, 31.9%). The post-thaw seminal ORP was negatively correlated with post-thaw TM% (r=-.5; p=.02), PM% (r=-.41; p=.03), TSC (r=-.60; p=.03) and CSR (r=-.52; p=.01). Increased levels of ORP are significantly correlated with poor post-thaw sperm quality and CSR.

Lyophilized seminal plasma can improve stallion semen freezability

The aim of this study was to evaluate the effect of lyophilized seminal plasma (LSP) on stallion semen freezability. Seminal plasma from 30 stallions was lyophilized to obtain a pool of LSP. Fifteen ejaculates from five stallions were supplemented before freezing with 0 mg/mL (Control), 1.44 mg/mL (LSP1), 5.04 mg/mL (LSP2) or 8.68 mg/ mL (LSP3) of LSP. Total antioxidant capacity (TAC) of LSP was assessed using Oxygen Radical Absorbance Capacity (ORAC) assay. Post-thaw motility and kinetics, sperm viability, normal morphology and membrane integrity were evaluated. Completely randomized mixed models were fitted for data analyses. The results was analyzed based on freezability of semen samples. TAC for LSP pool was 13679.4±911.6 μmol Trolox 100/g (ORAC units). Semen supplementation with LSP1 and LSP2 showed a positive effect on post-thaw total motility and membrane integrity. Supplementation with LSP3 showed a decrease in post-thaw total and progressive motility, straight line velocity and sperm viability. For poor freezability semen samples, supplementation with LSP1 and LSP2, showed higher post-thaw total motility and membrane integrity than good freezability semen samples. In conclusion, supplementation with LSP can improve the post-thaw seminal quality of stallion semen with poor freezability.

In vitro assessment of egg yolk-, soya bean lecithin- and liposome-based extenders for cryopreservation of dairy bull semen

The study was conducted to compare the effect of four commercially available extenders (Triladyl®- egg yolk-based; Andromed® and Bioxcell®-plant based and Optixcell®-liposome-based) on post‐thaw sperm quality and functionality variables evaluated using computer‐assisted sperm analysis and flow cytometry. A total of 30 ejaculates from five bulls were analysed. With use of Triladyl®, sperm had a greater post-thaw total motility than with use of Bioxell® and Optixcell® but there was no difference as compared with use of Andromed® with the greatest (P < 0.05) percentage of progressively motile cells. With use of Optixcell®, there was a greater (P < 0.05) percentage of sperm with an intact membrane than with use of Triladyl® and Bioxcell®, but values were similar with use of Andromed®. Acrosome damage in semen preserved with use of Optixcell® was less than with use of Bioxcell® and Andromed®. With use of Optixcell®, there was a greater percentage of viable spermatozoa with a lesser lipid disruption (P < 0.05) when compared with the other extenders. Production of peroxides was greater for sperm cryopreserved with use of Triladyl® and Optixcell® while less superoxide was produced in the samples cryopreserved with the egg yolk-based extender. Optixcell® appears to be a promising alternative to replace traditional egg yolk extenders. With use of Optixcell®, however, there were greater peroxide concentrations after thawing. With use of Andromed®, there were similar results as with use of Optixcell®, therefore, it could be an effective substitute for egg-yolk based media due to the greater proportion of highly and progressively motile spermatozoa at thawing.

Dimethylacetamide alone or in combination with glycerol can be used for cryopreservation of ovine semen.

Dimethylacetamide has been included in different extenders for the cryopreservation of semen from species with promising results. The objective of this study was to evaluate the use of dimethylacetamide (DMA) in different concentrations, associated or not with glycerol (GLY), for the cryopreservation of ovine semen, and its effects on in vitro sperm parameters and post-thaw in vivo fertility. Five semen samples of five adult Santa Ines sheep (n=25) were used. The collected ejaculates were divided among the seven treatments for subsequent cryopreservation. The treatments presented different concentrations of DMA and GLY, being divided as G1: GLY 6%; G2: DMA 3%; G3: GLY 5% + DMA 1%; G4: GLY 4% + DMA 2%; G5: GLY 3% + DMA 3%; G6: GLY 2% + DMA 4%; G7: GLY 1% + DMA 5%. %. Post-thawing of the straws, aliquots were evaluated for computerized sperm kinetics (CASA) and plasma membrane integrity, using fluorescent probes and flow cytometry. After the in vitro evaluation of the sperm parameters, in vivo testing was performed by laparoscopic artificial insemination of 72 females. The post-thaw total motility (%) evaluated by CASA were 51.4, 51.4, 50.1, 53.6, 52.3, 52.8 and 46.9, respectively, for the seven groups. And the plasma membrane integrity (%) were 19.7, 28.4, 22.3, 29.4, 24.3, 17.9 and 16.9, respectively. There were no differences (P> 0.05) between the treatments for the parameters of spermatic kinetics and membrane integrity. For females inseminated with semen from the control group (G1, GLY6%), the percentage of pregnant females was 36.1%, a result similar to that obtained with G3 treatment (GLY5% + DMA1%). In conclusion, dimethylacetamide, either alone or in combination with glycerol, can be used for cryopreservation of ovine semen.

Effects of Different Freezing Protocols on Motility, Viability, Mitochondrial Membrane Potential, Intracellular Calcium Level, and DNA Integrity of Cryopreserved Equine Epididymal Sperm

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous.

Increased cryo-survival rate in ejaculated human sperm from infertile men following pre-freeze in vitro myo-inositol supplementation.

ObjectiveTo investigate the effects of in vitro myo-inositol (Myo-Ins) supplementation of cryopreserved human semen on the cryo-survival rate (CSR).MethodsSemen samples were obtained from 41 infertile men. Following routine semen analysis, each sample was divided into two equal aliquots (0.5 mL each). One aliquot was treated with 1 mg of Myo-Ins dissolved in 10 µL of sperm preparation medium. The second aliquot was treated with 10 µL of the same medium (control). Both aliquots were incubated for 20 minutes prior to freezing to slow the freezing process. The frozen samples were examined for post-thaw percentages of total motility (TM), progressive motility (PM), and the CSR, defined as the percentage of post-thaw TM divided by the percentage of pre-freeze TM and multiplied in 100. The results were expressed as median and interquartile range (25th and 75th percentiles).ResultsThe pre-freeze TM (50% [30%–50%]) and PM (35% [20%–35%]) were significantly higher than the post-thaw TM and PM in the Myo-Ins group (15% [10%–35%] and 10% [5%–20%]; p<0.001 and p<0.001, respectively) and the control group (10% [6%–30%] and 5% [3%–15%]; p<0.001 and p<0.001, respectively). The CSR of the 41 semen aliquots supplemented with Myo-Ins (40% [25%–70%]) was significantly higher than that of the control samples (30% [13%–58%], p=0.041). The CSR of the 26 abnormal semen samples that were supplemented with Myo-Ins (38% [20%–50%]) was significantly higher than that of the control samples (23% [12%–30%], p=0.031).ConclusionIn vitro Myo-Ins supplementation of ejaculated human sperm from infertile men resulted in a significant increase in the CSR in samples with abnormal pre-freeze sperm parameters.

Improving cryopreservation capacity of ram spermatozoa by supplementing the diluent with melatonin

The aim of this investigation was to evaluate the efficiency of supplementing ram semen extender with melatonin on cryopreservation capacity of spermatozoa. A total of 80 ejaculates were collected from 5 Barki rams, 16 ejaculates each, by an artificial vagina throughout the period from January – February, 2017. Ejaculates of each collection session were pooled and diluted (1:10) with glycerolated Tris-citric egg yolk extender, and were split into 4 portions. The first portion served as control (melatonin-free), whereas the other 3 portions were supplemented with 0.1, 0.2 or 0.3 mM melatonin. The post-thaw objective assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer-assisted sperm analysis (CASA) system. The results revealed that melatonin supplementation was positively correlated (P<0.01) with post-thaw total motility, progressive motility, viability, acrosome integrity, sperm cell membrane integrity, VSL, VAP and WOB (r= 0.88, 0.71, 0.78, 0.85, 0.96, 0.95, 0.68, 0.57 and 0.53, respectively. However, it was negatively correlated (P<0.01) with primary and secondary sperm abnormalities, as well as non-progressive motility and immotile spermatozoa (r= -0.73, -0.85, -0.49 and -0.76, respectively. These results elucidate that adding melatonin to ram semen extender substantially enhanced post-thaw sperm physical properties, which implies its powerful potential as an exogenous antioxidant supplement.