Post-thaw Semen Parameters Research Articles

Association of Endogenous Seminal L-Carnitine Levels with Post-Thaw Semen Parameters in Humans

Cryopreservation of semen is a useful tool for male fertility preservation. Some evidence for a beneficial effect of L-carnitine supplementation of freezing media on cryopreserved semen samples has been reported. Here, we examined the association of endogenous levels of seminal L-carnitine with post-thaw semen parameters. We also investigated the effect of freezing medium supplemented with L-carnitine on sperm characteristics, related to endogenous seminal L-carnitine levels. Semen analyses were performed on 125 fresh samples, and after standard cryopreservation and with L-carnitine as a supplement. Participants were categorized into two groups based on the median levels of endogenous seminal L-carnitine: low L-carnitine (≤38.8 µg/ml) and high L-carnitine (>38.8 µg/ml). After standard cryopreservation, semen samples with high L-carnitine levels showed higher rapid progressive, progressive and total sperm motility and a reduced seminal static oxidation–reduction potential (ORP) level than samples with low L-carnitine levels. Only in post-thaw samples with low L-carnitine levels, there was an increase in the amount of sperm neck midpiece defects, compared to the fresh samples. Cryopreservation with L-carnitine had the most beneficial effect on rapid progressive sperm motility in samples with high endogenous L-carnitine levels. In conclusion, L-carnitine has a beneficial impact on sperm characteristics in post-thaw samples both as an endogenous component in seminal plasma and as a supplement in the freezing medium, by improving sperm motility and reducing seminal oxidative stress.

The optimum concentration of soya-lecithin for replacing egg yolk in INRA-82-modified semen extenders improved the cryopreserved semen quality and DNA integrity of Arabian stallions

The cryopreservation of stallion spermatozoa varies greatly between animals. Different equine semen extender supplements were invented to maximize the post-thaw semen quality. Soy lecithin is one of those additives that contain plant origin phospholipids used to replace egg yolk to minimize disease transmission. This study aimed to determine which concentrations of soy lecithin can replace egg-yolk in modified INRA-82 semen extenders for the cryopreservation of horse semen. Semen was collected from six Arabian stallions and extended with modified INRA-82 semen extender supplemented with 0.0, 1.25, 2.50, and 5.0% soya lecithin. Frozen-thawed sperm motility was measured at 0, 1, 2, and 3 h. Viability index, sperm plasma membrane integrity (SPMI), and acrosome integrity in addition to the comet assay non-fragmented DNA, head DNA, tail DNA, tail length, and tail moment were statistically analyzed. The addition of 2.5% soya lecithin had the highest post-thaw sperm motility at 0, 1, 2 h (P < 0.001) and 3 h (P < 0.0001), the optimum viability index (P < 0.0001), SPMI (P < 0.05), acrosome integrity (P < 0.0001), non-fragmented DNA (P < 0.01), DNA in the comet head (P < 0.05), minimum DNA in the comet tail (P < 0.05), shortest comet tail length (P < 0.0001), and lowest comet tail moment (P < 0.01). Soy lecithin at concentrations other than 2.5% deteriorated all post-thaw semen parameters. Soy lecithin (2.5%) can replace egg yolk in the modified INRA-82 semen extender for the cryopreservation of good-quality stallion semen.

Mito-Tempo improves acrosome integrity of frozen-thawed epididymal spermatozoa in tomcats.

In tomcats, epididymal spermatozoa provide an additional source of male gametes available for cryopreservation. While this procedure is feasible, the survival rate and motility of epididymal cat spermatozoa are both low after thawing. Cryopreservation is known to induce oxidative stress in spermatozoa, with mitochondria and the plasma membrane being the two major generation sites, and an imbalanced presence of free radicals is a possible cause for this low survival rate. Different antioxidants have been tested before for their effect on cryopreserved cat spermatozoa quality, with varying results. Here, we used Mito-Tempo, which is a synthetic mitochondria-targeted antioxidant and a specific scavenger of the mitochondrial superoxide system. By supplementing Mito-Tempo with the freezing extender, we aimed to improve the sperm quality of frozen-thawed cat epididymal spermatozoa. Epididymal spermatozoa obtained from twelve tomcats were assessed for motility and concentration. Prior to freezing, samples were diluted in TRIS buffered extender with egg yolk and glycerol and divided into five aliquots supplemented with 0 (control), 0.5, 5, 50, and 1005M of Mito-Tempo. After thawing, sperm motility, concentration, morphology, plasma membrane integrity, acrosome integrity, and mitochondrial membrane potential were evaluated. A Friedman rank sum test with a Bonferroni post-hoc test was used to determine statistical in-between group differences in post-thaw semen parameters. The results indicated a slight improvement in acrosome integrity across all groups that were supplemented with Mito-Tempo, with the group that received 55M of Mito-Tempo showing the greatest improvement [(median of 67.99%, IQR of 5.55) compared to the control group (median of 65.33%, IQR of 7.75; P = 0.05)]. For all other sperm parameters, no significant differences (P > 0.05) were detected between different Mito-Tempo concentrations. These findings highlight the protective effect of Mito-Tempo on acrosome integrity and suggest that 55M is the most effective concentration for maintaining acrosome integrity. Since Mito-Tempo has shown a positive effect on multiple sperm parameters in other species, such as men, boars, roosters, rams, and bulls, we need to conclude that species-specificity may play a role here.

Hydroxytyrosol and resveratrol improves kinetic and flow cytometric parameters of cryopreserved bull semen with low cryotolerance

There is considerable interest in breeding programs to “rescue” semen with poor post-thaw fertility from bulls known as “bad freezers”. We hypothesized that there may be an interaction between the post-thaw recovery of sperm and the efficacy of antioxidant addition to extenders. The current study assesses the effects of antioxidant additives hydroxytyrosol (HT) and resveratrol (RSV) on the post-thaw semen parameters in two groups of bulls classified as either high or low cryotolerant (i.e., “good” and “bad” freezers). Semen samples were collected from 40 bulls and processed in the extenders containing different concentrations of HT (experiment 1; 0, 25 and 50 µM) and RSV (experiment 2; 0.0, 0.01, 0.1 and 1 mM). In experiment 1, bulls in the low cryotolerance group had a significant improvement in post-thaw recovery at 25 µM and 50 µM (P < 0.05). These improvements were observed in motility and several cellular parameters. However, post-thaw semen quality in the high cryotolerance group was not significantly affected by the HT addition. In experiment 2, although RSV did not have any positive impact in high cryotolerance group, post-thaw recovery in the low cryotolerance bulls was significantly improved in 0.1 mM RSV. Oxidative stress markers in either high or low cryotolerance groups were not significantly changed by RSV addition (P > 0.05). It can be concluded that addition of optimized concentrations of HT and RSV to the extender could be a strategy for improving the post-thaw semen, especially in bulls with high genetic merit but low initial cryotolerance.

Effect of Betaine and Raffinose in Cryopreservation Medium on Fertility in Kadaknath Chicken

BACKGROUND: Kadaknath is an important indigenous chicken with black pigmentation and cryopreserved semen reputably had low fertility. OBJECTIVE: The aim of this study was to evaluate the effects of betaine and raffinose in semen extenders on post thaw semen parameters and fertility. MATERIALS AND METHODS: Semen was cryopreserved in 4% dimethyl sulfoxide (DMSO) with betaine supplemented at 0.1, 0.2 and 0.4 M or raffinose supplemented at 1, 5 and 10 mM. Post thaw semen parameters and fertility were evaluated. RESULTS: Betaine at higher concentrations significantly (p &lt; 0.05) inhibited the post thaw sperm motility, live sperm and MTT dye reduction and a declining trend in the fertility with increasing betaine. Inclusion of raffinose had no effect on the post thaw in vitro semen parameters, however, the fertility was significantly (p &lt; 0.05) higher in the 10 mM raffinose supplemented group. CONCLUSION: Betaine has negative effect on post thaw semen parameters and raffinose at 10 mM concentration improves the fertility from cryopreserved semen.

Effect of zinc supplementation during cryopreservation on post-thaw chicken semen parameters and fertility

The present study evaluated supplementation of zinc from different sources during chicken semen cryopreservation on post-thaw semen quality and fertility. Adult White Leghorn chicken semen was cryopreserved using 4% dimethyl sulfoxide (DMSO) in Sasaki diluent (SD). In the semen cryomixture zinc oxide (6.25 and 12.5 µM) and zinc sulphate (100 and 200 µM) was added and cryopreserved. The plastic semen straws were thawed at 5°C for 100 sec and samples were evaluated for sperm motility, live, abnormal and acrosome intact sperm. The lipid peroxidation in the seminal plasma was evaluated. The semen cryopreservation and evaluation were repeated on six occasions. Fertility of the zinc supplemented semen was evaluated by inseminating into White Leghorn hens. Zinc supplementation did not affect any of the post-thaw sperm parameters. The lipid peroxidation and fertility were similar between the treatments. In conclusion, supplementation of zinc as zinc oxide or zinc sulphate during chicken semen cryopreservation does not affect post-thaw semen parameters or fertility.

P–441 Semen quality and cryopreservation in adolescent transgender females

Abstract Study question What are the semen quality and cryopreservation outcomes among adolescent transgender females at the time of fertility preservation (FP) before initiating gender-affirming hormone (GAH) treatment? Summary answer Semen quality is strongly reduced among adolescent transgender females before hormone therapy and their stored sperm samples are suitable for intracytoplasmic sperm injection (ICSI). What is known already The age of individuals seeking treatment for gender affirmation has fallen sharply in recent years and many of them are adolescents. Estrogen, the primary treatment for transgender women, is known to impair semen quality and fertility potential. Sperm cryopreservation enables young transgender females to circumvent GAH therapy-related fertility impairment and have genetically related children. There are recent data on semen quality among adult transgender women who preserve fertility before exposure to GAH therapy, but little is known about pubertal transgender female adolescents. Study design, size, duration This retrospective cohort study included 26 adolescent transgender females who underwent FP between June 2013 and October 2020. Participants/materials, setting, methods Before initiating gonadotropin-releasing hormone agonists solely or with GAH treatment, 25 adolescent transgender females were referred to FP in our Fertility Institute of a tertiary university-affiliated medical center. Pre-freezing semen parameters were compared to WHO 2010 reference values. Post-thaw semen parameters were used to determine adequate assisted reproductive technology (ART). A multivariate linear regression analysis was performed to assess the impact of selected medical and lifestyle factors on the semen quality of our study participants. Main results and the role of chance The mean age at which adolescent transgender females underwent sperm cryopreservation was 16.2 ± 1.38 years. The median values of all semen parameters in our study group were significantly lower compared to the WHO data on semen quality in the general population of unscreened men, including volume (1.46 ml vs 3.2 ml, respectively, P = 0.001 ), sperm concertation (28*106/ml vs 64*106/ml, P &amp;lt; 0.001), total sperm number (28.2*106 vs 196*106, P &amp;lt; 0.001), total motility (51.6% vs 62%, P &amp;lt; 0.001), and normal morphology (2% vs 14%, P &amp;lt; 0.001). The frequency of semen abnormalities was teratozoospermia 72%, hypospermia 52%, oligozoospermia 28%, and azoospermia 4%. The median post-thaw total motile count was 0.17*106 per vial, and the quality was adequate only for ICSI in 87.7% of the thawed semen samples. Attention-deficit/hyperactivity disorder (ADHD) diagnosis, history of depression/anxiety, medication for ADHD, and antidepressant drugs were found to correlate with hypospermia. No correlation was found between the time of FP, body mass index, autistic spectrum disorder diagnosis, cannabis use, testis tucking, or the levels of follicle-stimulating hormone, estradiol, and testosterone on the semen parameters. Limitations, reasons for caution Because no normal values of semen in adolescents are available and the absence of a matched control group, we used WHO 2010 semen data as reference values, and they may not be representative of the adolescent population. Wider implications of the findings: Although adolescent transgender females have poor semen quality and limited stored semen samples suitable for advanced ART interventions, even before starting GAH therapy, we highly recommend sperm cryopreservation before initiating GAH treatment and thereby prevent further impairment of sperm quality associated with the hormonal treatment. Trial registration number Not applicable

Adolescent Transgender Females Present Impaired Semen Quality That Is Suitable for Intracytoplasmic Sperm Injection Even Before Initiating Gender-Affirming Hormone Treatment

The present study aimed to determine the semen quality and cryopreservation outcomes among adolescent transgender females at the time of fertility preservation (FP) before initiating gender-affirming hormone (GAH) treatment. This retrospective cohort study included 26 adolescent transgender females who underwent FP in our Fertility Institute between 06/2013 and 10/2020. Pre-freezing semen parameters were compared to WHO 2010 reference values. Post-thaw semen parameters were used to determine the adequate assisted reproductive technology (ART). A multivariate linear regression analysis was performed to assess the impact of medical and lifestyle factors on semen quality. The mean age at which adolescent transgender females underwent FP was 16.2 ± 1.38 years. The median values of all semen parameters in our study group were significantly lower compared to the WHO data, including volume (1.46 mL vs 3.2 mL, respectively, P = 0.001 ), sperm concentration (28 × 106/mL vs 64 × 106/mL, P < 0.001), total sperm number (28.2 × 106 vs 196 × 106, P < 0.001), total motility (51.6% vs 62%, P < 0.001), and normal morphology (2% vs 14%, P < 0.001). The frequency of semen abnormalities was teratozoospermia 72%, hypospermia 52%, oligozoospermia 28%, and azoospermia 4%. The median post-thaw total motile count was 0.17 × 106/vial, and the quality was adequate only for ICSI in 87.7% of the thawed semen samples. No correlation was found between selected medical and lifestyle factors and poor semen parameters. Semen quality is strongly reduced among adolescent transgender females before hormone therapy and their stored sperm samples are suitable for intracytoplasmic sperm injection (ICSI) rather than conventional IVF/intrauterine insemination (IUI).

High or Low Body Fat Deposition in the Presence of a Normal Oral Sugar Test is Not Associated With Postthaw Semen Parameters in Stallions

This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallions. One ejaculate from each stallion was frozen with a commercial egg yolk-based extender. Postthaw sperm motility parameters, plasma membrane integrity, mitochondrial membrane potential, hydrogen peroxide and intracellular superoxide production, and lipid peroxidation were analyzed for all stallions. The circumference at 25% and 50% of the neck’s length were larger for High-BCS stallions (P < .05). There were no differences between groups for the neck crest height (P > .05). Stallions with High BCS had greater SFT at the tail head than stallions with Low BCS (P < .05); however, there were no differences between groups in the SFT at the shoulders and withers (P > .05). All stallions had resting blood glucose below the cutoff for equine metabolic syndrome. There were no differences between groups for resting glucose concentrations or for a peak at 30 or 60 minutes after initiation of the OST (P > .05). There were no differences in sperm parameters between groups (P > .05). Collectively, the findings of the present study suggest that High BCS or Low BCS in the presence of normal OST do not explain post-thaw semen parameters.

Impaired semen quality in trans women: prevalence and determinants.

STUDY QUESTIONWhat is the semen quality in trans women at time of fertility preservation, prior to the start of gender-affirming hormone treatment?SUMMARY ANSWERBefore the start of gender-affirming hormone treatment, semen quality in trans women was already strongly decreased compared to the general population.WHAT IS KNOWN ALREADYHormone treatment for -trans women (birth-assigned males, female gender identity) consists of anti-androgens combined with estrogens in order to achieve feminization and it is accompanied by a loss of reproductive capability. Trans women can opt for semen cryopreservation prior to their medical transition to retain the possibility to parent genetically related offspring. Post-thaw semen parameters determine which ART can be used. Knowledge of semen quality and the factors negatively influencing semen parameters in trans women are important to improve semen quality before fertility preservation.STUDY DESIGN, SIZE, DURATIONA retrospective cohort study was performed between 1972 and 2017. In total, 260 trans women were included for this study. Due to the study design, there was no loss to follow-up or attrition.PARTICIPANTS/MATERIALS, SETTING, METHODSWe studied the quality of the preserved semen in trans women, prior to their medical transition, who visited our gender clinic. Semen parameters were collected, as well as data on age, alcohol consumption, smoking, cannabis use, BMI, previous use of estrogens or anti-androgens and endocrine laboratory results. Semen parameters were categorized using reference values for human semen of the World Health Organization (WHO) and compared with data from the general population. Logistic regression analyses were performed to analyze the extent to which factors known to have a negative impact on semen quality in the general population explained the impaired semen quality in the cohort.MAIN RESULTS AND THE ROLE OF CHANCEThe cohort consisted of 260 trans women between the age of 16 and 52 years. Semen quality in trans women was significantly decreased compared to WHO data from the general population. In total, 21 trans women had an azoospermia and median semen parameters for the remaining trans women and the general population, respectively, were as follows: volume 2.7 and 3.2 ml (P < 0.05), sperm concentration 40 and 64 million/ml (P < 0.05), total sperm number 103 and 196 million (P < 0.05) and progressive motility 41% and 57% (P < 0.05). Smoking (odds ratio (OR) 2.35 (95% CI 1.06–5.21)) and a higher age at time of fertility preservation (OR 1.04 (95% CI 1.00–1.08)) were found to correlate with an impaired progressive motility. Twelve trans women reported to have used anti-androgens and estrogens, and all had discontinued for at least 3 months prior to the first attempt for semen cryopreservation. No correlation was found between previous gender-affirming hormone use and decreased semen parameters. The median post-thaw total motile sperm count was 1.0 million per vial (interquartile range 0.1–3.1) and in only 26.4% of thawed semen samples was the quality adequate for a minimally invasive IUI.LIMITATIONS, REASONS FOR CAUTIONLimitations include the retrospective design and insufficient data on transgender-specific factors, such as bringing the testes into the inguinal position (tucking), wearing tight underwear and low masturbation frequency.WIDER IMPLICATIONS OF THE FINDINGSSemen quality in trans women was decreased compared to the general population, which could not be explained by known risk factors, such as BMI, alcohol consumption, cannabis use, gender-affirming hormone use or abnormal endocrine laboratory results. Although a negative impact of smoking was observed, it was insufficient to explain the overall decreased semen quality in this cohort. Since low pre-freeze semen quality results in an even lower post-thaw semen quality, the majority of trans women and their female partner or surrogate may need an invasive and burdensome treatment to establish a pregnancy.STUDY FUNDING/COMPETING INTEREST(S)For this study, no external funding was obtained and there were no competing interests.TRIAL REGISTRATION NUMBERNA.

Addition of pomegranate juice (Punica granatum) in tris-based extender improves post-thaw quality, motion dynamics and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa.

The aim of the present study was to determine the protective effects of pomegranate juice in tris-based extender on semen parameters, computer-assisted sperm analysis (CASA) motion characteristics and field fertility of post-thawed Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Two consecutive ejaculates/collection from each of the five adult Nili Ravi buffalo bulls were collected with artificial vagina at 42°C for a period of 7weeks, diluted in extender containing different concentrations of pomegranate juice (0.0%, 2.5%, 5%, 7.5% and 10%). Diluted samples were packed and frozen in 0.54ml French straws. The addition of 10% pomegranate juice in extender significantly improved post-thaw sperm morphology (%), motilities (CASA total motility, progressive motility (%) as well as VAP, VSL, VCL, STR, DAP, DSL) compared to the control group (p<0.05). Plasma membrane, acrosome membrane and DNA integrity were significantly higher in extender with 10% pomegranate juice than the control group (p<0.05). Field fertility rate (60.39% vs. 46.53%) was higher (p<0.05) in extender with 10% pomegranate juice as compared to the control. It is therefore concluded that the addition of 10% pomegranate juice in tris-based extender improves post-thaw semen parameters, CASA motion dynamics and field fertility in Nili Ravi buffaloes.

Effect of supplementing vitamin E analogues on post-thaw semen parameters and fertility in chicken

ABSTRACT 1. The effect of supplementing water-soluble vitamin E analogues 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) and butylated hydroxy toluene (BHT) was studied in two separate experiments. 2. In the first experiment, trolox was supplemented at 0.2 mM, 0.4 mM and 0.8 mM concentrations along with N-methylacetamide (MA; 12% final concentration) and semen was cryopreserved in 0.5 ml French straws. Different semen parameters and fertility were assessed from post-thaw samples. 3. Sperm motility, live sperm, and mitochondrial activity were significantly lower (P < 0.05) in cryopreserved semen. Lipid peroxidation (LPO) was significantly higher (P < 0.05) in cryopreserved semen that was reduced by trolox supplementation. The treatment containing trolox at 0.2 mM concentration produced significantly higher (P < 0.05) fertility compared to unsupplemented cryopreservation treatment. 4. In the second experiment, BHT was supplemented at 0.25 mM, 0.5 mM, and 1 mM concentrations along with MA during semen cryopreservation. 5. Sperm motility, live sperm and MTT dye reduction test were significantly lower (P < 0.05) in cryopreserved semen. These parameters declined with increasing BHT concentration. Abnormal sperm was significantly higher (P < 0.05) in the BHT supplemented treatments. The sperm chromatin dispersion (SCD) test was significantly higher (P < 0.05) in cryopreserved samples and was highest in samples supplemented with 0.5 mM and 1 mM BHT. The percentage fertility was significantly lower (P < 0.05) in cryopreserved semen and BHT supplementation did not improve fertility. 6. In conclusion, trolox supplementation at 0.2 mM concentration during semen cryopreservation improved fertility, whereas BHT supplementation resulted in a decline in post-thaw semen parameters.

Cryoprotective role of organic Zn and Cu supplementation in goats (Capra hircus) diet

The current study focused on cryopreservation and assessment of characters of post-thaw semen of indigenous Osmanabadi bucks maintained with standard diet, supplemented with different concentrations of organic zinc (Zn), copper (Cu) or in combination, for a period of 180 days. The different doses of organic Zn and Cu were fed per kg DM basis, Zn groups (low: Zn20, medium: Zn40 and high: Zn60), Cu groups: (low: Cu12.5, medium: Cu25 and high: Cu37.5) and combination of Zn + Cu groups (low: Zn20 + Cu12.5, medium: Zn40 + Cu25 and high: Zn60 + Cu37.5) respectively. The control group bucks were maintained mainly on the basal diet without any additional mineral supplementation. Two hundred and forty (240) semen samples were collected from 40 bucks aged 11 months, through electro ejaculator method, processed and analysed for sperm quality parameters both at pre freeze and post-thaw stage. The semen samples were diluted in Tris egg yolk extender, cooled and equilibrated for 4 h at 5 °C, cryopreserved using programmable freezer (PLANER Kryo 360–1.7) and stored at −196 °C. The organic trace minerals (Zn, Cu and Zn + Cu) protected the spermatozoa against the cryoinjury and maintained higher post-thaw semen parameters except in high Zn group. Additional feeding of organic Cu and Zn to bucks had a protective role and resulted in higher sperm liveability, plasma membrane and acrosome integrities, motility and velocity and reduced oxidative stress in supplemented goats (P < 0.05).

A retrospective study on sperm banking: a Uruguayan experience.

The purpose of this study was to investigate the status of homologous sperm banking in Uruguay. A retrospective investigation was performed on data collected between 2013 and 2015. Reasons for sperm banking, patient age, pre-freeze and post-thaw semen parameters, and recovery rates were analyzed. 623 samples were cryobanked between 2013 and 2015. Only 324 samples were considered for analysis after selection based on inclusion criteria. In most cases the samples were stored because the patients were undergoing assisted reproductive technology (ART) treatment (n=190; 58,64%) or for oncological reasons (n=113; 34,88%). The median age of bankers was 34 years. In the cancer group, 61.95% (n=70) of the subjects had been diagnosed with testicular cancer. Medians of semen parameters for both groups were above the lower reference limits dictated by the . In fresh samples, a significant difference was observed in progressive motility (47% vs. 56%) between ART and oncological patients. After thawing, total motility (27% vs. 32%), progressive motility (19% vs. 22%), and vitality (48% vs. 56%) differed significantly between ART and oncological bankers. Semen banking has been performed successfully in Uruguay and outcomes are on par with international standards. Surprisingly, the semen parameters of the cancer group were nearly normal.

Effect of Seminal Plasma Components on the Quality of Fresh and Cryopreserved Stallion Semen

The importance of seminal plasma (SP) components for stallion semen quality and freezability is little known. This study aimed to evaluate the relationship between SP components and fresh/cryopreserved stallion semen quality. Semen of 30 stallions was collected, and then, SP was recovered and lyophilized. Total protein (TP), vitamin C (CVIT), vitamin E (EVIT), vitamin A (AVIT), iron (Fe), copper (Cu), magnesium, and zinc (Zn) in SP were assessed. Sperm was frozen in an extender supplemented with lyophilized SP. In fresh semen motility, abnormal morphology (AM), sperm vitality (SV), and plasma membrane integrity (PMI) were evaluated. In post-thaw semen, additionally, total motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat cross-frequency (BCF) were assessed. Levels of component of SP were established by a distribution analysis. Generalized linear models were fitted. Comparisons of means were done with Tukey's test. Correlation and regression analyses were performed. Vitamins and ions were found to be related to fresh semen quality. For post-thaw sperm, medium TP showed higher semen quality. Negative regression and correlation coefficients between CVIT and all post-thaw semen parameters were found. Low EVIT yielded the lowest PM, VSL, and VAP values, while a high level of AVIT yielded the best results for sperm quality. A high level of Cu yielded higher results for TM, PM, VCL, and ALH. Moreover, a negative correlation was found between Zn, SV, and PMI. In conclusion, SP composition influences fresh and post-thaw stallion semen quality.

The Effects of an Oxygen Scavenger and Coconut Water on Equine Sperm Cryopreservation

Alternative sources of lipoproteins in semen extenders could replace animal by-products. We hypothesized that: (1) post-thaw semen parameters and fertility would not be different in coconut water (CW)–treated samples compared with egg yolk (EY)–treated samples and (2) the use of an oxygen scavenger (Oxyrase) would improve post-thaw sperm motility and membrane integrity and decrease lipid peroxidation. Experiment 1: three ejaculates each from five stallions were split into four treatments: EY, CW, egg yolk with Oxyrase, and coconut water with Oxyrase. Computer-assisted sperm analysis measured progressive and total motility, velocity, and linearity. Membrane integrity, apoptosis, and lipid peroxidation were evaluated using propidium iodide, annexin, and BODIPY fluorescent probes, respectively. Samples were cryopreserved, stored in liquid nitrogen, and then thawed to 37°C and analyzed again. Experiment 2: one ejaculate was divided into two aliquots and cryopreserved using either CW or EY. In a crossover design, 12 mares were bred on two consecutive cycles with either EY or CW. Pregnancy evaluations were at 14-day gestation. No differences were detected in sperm parameters between CW and EY (P > .05). Oxyrase did not improve sperm motility parameters in post-thaw samples, nor did it show protective effects for viability or against membrane damage (P > .05). More mares became pregnant using CW than EY (11/12 vs. 6/12, respectively; P = .013). Use of CW is a viable alternative to animal-based products in the cryopreservation of stallion semen.

Effects of Repeated Partial Thaw and Refreeze on Post-Thaw Parameters of Stallion Semen Cryopreserved in Cryovials

The current study evaluated post-thaw semen parameters of stallion semen cryopreserved in cryovials and subjected to multiple partial thaw-refreeze cycles. Five fertile stallions were collected twice, and ejaculates were analyzed for concentration, percent membrane integrity, motility, morphology, and sperm chromatin structure (SCSA). Semen processed with freezing extender from each ejaculate was cryopreserved in both 1.2-mL cryovials and 0.5-mL straws. Cryovials were subjected to eight subsequent partial thaw-refreeze cycles. Cryovials were warmed for approximately 30 seconds; then, a sample of cryopreserved semen was removed with a 16-gauge needle, and the cryovial was immediately refrozen in liquid nitrogen. A piece of 0.5-mL straw cut under liquid nitrogen from the same stallion and ejaculate was thawed alongside each cryovial to serve as a control. Thawed samples were analyzed for percent membrane integrity, motility, and SCSA. Post-thaw parameters of motility and membrane integrity were analyzed by one-way or two-way analysis of variance with repeated measures when appropriate. The SCSA data were analyzed using a mixed regression model. Post-thaw motility and percentage of intact sperm were significantly lower when sperm was cryopreserved in cryovials compared to straws. However, these parameters may remain adequate for use in assisted reproductive techniques (ARTs) such as intracytoplasmic sperm injection through all cryovial thaws. Additionally, DNA denaturability was not affected by semen packaging method and was only affected by thaw number, increasing at post-thaws 5 and 6. This technique may offer a unique approach for cryopreservation and utilization of stallion sperm for ARTs in the future.

Multivariate model for predicting semen cryopreservation outcomes in a human sperm bank.

Semen cryopreservation is widely used in assisted reproductive technologies, but it reduces sperm quality dramatically. The aim of this study was to develop a model using basal semen quality to predict the outcome of postthaw semen parameters and improve the efficiency of cryopreservation in a human sperm bank. Basal semen parameters of 180 samples were evaluated in the first stage, and a multiple logistic regression analysis involving a backward elimination selection procedure was applied to select independent predictors. After a comprehensive analysis of all results, we developed a new model to assess the freezability of sperm. Progressive motility (PR), straight-line velocity (VSL) and average path velocity (VAP) were included in our model. A greater area under the receiver operating characteristic curve was obtained in our model when compared with other indicators. In the second stage of our study, samples that satisfied the new model were selected to undergo freeze–thawing. Compared with the first stage, the rate of good freezability was increased significantly (94% vs 67%, P = 0.003). By determining basal semen quality, we have developed a new model to improve the efficiency of cryopreservation in a human sperm bank.

Incorporation of Ascorbic Acid, Caffeine and Chloroquine Diphosphate in Dilutor Improves Structural and Functional Status of Frozen Semen

Effect of supplementation of ascorbic acid, caffeine and chloroquine diphosphate in the dilutor on post-thaw quality of spermatozoa was studied. Thirty-two semen ejaculates (8 each from 2 Holstein Friesian and 2 crossbred; 50% Friesian x 50% Haryana) were studied. Ascorbic acid, caffeine and chloroquine diphosphate were added in Tris-egg yolk-glycerol (control) extender at the concentration of 10 mM, 0.54 mM and 7 mM, respectively. The post-thaw semen parameters studied were structural (acrosomal integrity and livability), functional [motility, per cent sperm responding to hypoosmotic swelling test, i.e. HOS positive sperm and penetrability of sperm in bovine cervical mucus, i.e. sperm penetration distance-SPD] and measurement of activities of Glutamic oxaloacetic transaminase (GOT), Glutamic pyruvic transaminase (GPT), Alkaline transaminase (AKP) and Acid transaminase (ACP). Certain enzymes in the seminal plasma. The semen from crossbred bull had significantly lower mass activity (P ≤ 0.01), initial progressive motility (P ≤ 0.01), sperm penetration distance (SPD value) (P ≤ 0.01), HOS positive sperm and seminal plasma GOT activity, but higher abnormal sperm. Incorporation of additives in the dilutor significantly improved the post-thaw semen quality, especially the post-thaw motility, post-thaw per cent live sperm, sperm penetration distance and sperm responding to hypo-osmotic solution. The leakage of all the 4 enzymes (GOT, GPT, ACP and AKP) was significantly less in the samples frozen in presence of ascorbic acid and chloroquine as compared to control. Ascorbic acid resulted in maximum improvement to the quality of frozen-thawed semen followed by chloroquine and caffeine. The study concludes that post-thaw quality of frozen semen of HF and crossbred bulls significantly improves if the dilutor is supplemented with 10 mM concentration of ascorbic acid.

Predictors of sperm recovery after cryopreservation among men with myeloid leukemia (ML) and non-hodgkin's lymphoma (NHL)

To identify modifiable factors in cryopreservation protocols for the optimal sperm recovery in with ML and NHL, a cohort previously identified as having a significant decrease in post thaw semen parameters.