Effects of semen quality on pregnancy rate in artificially inseminated dairy cows

Oxidative stress has been identified as a major cause of low seminal fertility. Among the components of stallion seminal plasma, some enzymatic and non-enzymatic antioxidants have been identified, which protect sperm from injurious effects of reactive oxygen species (ROS); however, the characterisation of these components is still in preliminary stages, as well as their relationship with freezability. The aim of this study was to evaluate the effect of some components of seminal plasma (SP) on stallion semen freezability. Semen of 30 Colombian Creole horses, and a total of 60 ejaculates, were collected. Semen was centrifuged to recover the SP. It was lyophilized and some components were assayed: total protein concentration (TP) by Bradford assay, CRISP3 protein concentration by ELISA, vitamin C (CVIT), vitamin E (EVIT) and vitamin A (AVIT), by HPLC; content of iron (Fe), copper (Cu), magnesium (Mg) and Zinc (Zn) by atomic absorption spectroscopy flame. Semen was supplemented with 10% stallion lyophilized SP and cryopreservation was performed. Post-thaw, 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 using a computer-assisted sperm analysis (SCA®, Microptic SL, Barcelona, Spain). Sperm viability (SV) was determined by the Live/Dead kit (Molecular Probes Inc., Eugene, OR, USA). Normal sperm morphology (NM) was performed by the supravital technique and plasmatic membrane integrity (MI) was evaluated by the hypo-osmotic test. For statistical analysis, completely randomised mixed models were fitted. Levels according to the concentration of components of SP (high, medium, and low) were established. Comparisons of the means between levels were done with Tukey’s test. The significance level used for all assessments was P < 0.05. Means for TP of 0.35 mg BSA/g, CRISP3 of 55.22 ng/mg, CVIT of 2.66 mg/g, EVIT of 72.36 µg/g, AVIT of 37.37 µg/g, Fe of 17.37 mg/kg, Cu of 33.64 mg/kg, Mg of 109.08 mg/kg, and Zn of 0.49 g/100 g of SP were found. We found that a high level of CRISP3, AVIT, Cu, and Fe had higher results for post-thaw TM, PM, NM; medium levels of TP and Mg showed higher post-thaw TM, PM, NM, and MI; and lower levels of Zn had better results for post-thaw TM, PM, VCL, and VAP. In contrast, high and medium levels of CVIT had a deleterious effect on post-thaw TM, PM, SV, NM, and MI. We concluded that there is a relationship between concentrations of seminal plasma components and stallion semen freezability.

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

Buffalo semen collected from Murrah bull were cryopreserved and evaluated for different motility parameter, kinematics and plasma membrane integrity. Buffalo bulls were maintained uniform standard management and nutritional practices. Semen was collected regularly twice a week semen collection schedule from four (04) Murrah bull. Collected semen was immediately transported to laboratory and evaluated for different macroscopic parameter (color, volume and thickness). Fresh semen was then diluted with saline solution and evaluated for sperm concentration, motility, sperm kinematics and morphology. Semen samples that fill all the standard were selected for freezing and diluted with Tris-egg yolk citrate diluter. Diluted semen was equilibrated, cryopreserved and finally evaluated for post thaw sperm quality. Different motility parameter (total, progressive, static and slow motility) varied significantly (p<0.01) irrespective of different freezing stages. Significantly higher progressive sperm motility and viability of buffalo spermatozoa were observed at fresh semen whereas lower progressive sperm motility and viability was found at post thaw stage. Total and progressive motility reduced by 2.5 and 2.12% following equilibration, whereas following cryopreservation, total and progressive motility reduced by 35.7 and 28.51% and static motility increases accordingly (35.4%). Significantly higher plasma membrane integrity of sperm was observed at fresh semen followed by pre freeze and post thaw semen. Following freezing, integrity of plasma membrane reduces at the rate of 10.81% and 26.7% at pre freezing and post thaw stages. Significantly higher average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were found for fresh semen followed by pre-freeze and post-thaw semen. Frozen buffalo semen with higher progressive motility and motion characteristics may be produced if motility losses can be reduced during freezing stage as this stage results higher motility losses. Asian Australas. J. Biosci. Biotechnol. 2022, 7(2), 75-81

Sixty semen ejaculates from 10 mature bulls, 5 each of Jafarabadi and Mehsana breed, were studied for sperm motility and velocity parameters of fresh and frozen-thawed spermatozoa using computer assisted sperm analyzer (CASA). The mean values of motile and progressively motile spermatozoa observed in fresh semen of Jafarabadi and Mehsana bulls (79.77±1.62 and 61.80±1.85, and 78.90±1.22 and 61.37±1.58%) were highly significantly (Pand medium motile sperm (%) increased, while beat-cross frequency (Hz), straightness (%), linearity (%), sperm area (µm2) and rapidly motile sperm (%) decreased significantly in post-thawed sperms when compared with the fresh sperm of both Jafarabadi and Mehsana bulls. The initial motility and live sperm per cent were significantly correlated with CASA traits of fresh and frozen-thawed semen, and all the sperm motility and velocity traits of fresh and frozen-thawed semen assessed by CASA were significantly interrelated among both the breeds. The interrelationships were stronger in Mehsana bulls as compared to Jafarabadi bulls.

This investigation was carried out on semen of three healthy mature breeding bulls of Gir breed to evaluate the interrelationships among sperm quality attributes of fresh and frozen-thawed semen assessed by Biovis CASA. The ejaculates (n = 24) having >75% initial motility were diluted @80 million sperm/mL using TFYG extender, filled in French mini straws, and were frozen using a programmable bio freezer after 4 hours of equilibration. The straws were thawed in a water bath at 37°C for 30 sec. The freshly diluted and frozenthawed samples were assessed for routine subjective tests and various motion characteristics/kinematics by Biovis CASA. The Pearson’s correlations for sperm motility and velocity/kinematic parameters of total motile sperm as well as of progressively motile sperm were studied in freshly diluted and frozen-thawed semen. In fresh semen, total motile sperm assessed by CASA had significant (p less than 0.05, 01) correlations with rapid progressive motile sperm (r = 0.46), wobbling index (r = 0.52) and dancing frequency (r = -0.43) in fresh semen. In frozen-thawed semen, it was significantly correlated only with linearity (r = 0.46). The rapid progressive motile sperm in both fresh (r = 0.41 to 0.92) and frozen-thawed (r = 044 to 0.88) semen, however, had significant correlations with most of their velocity traits. Further, the average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN), straightness (STR), wobbling (WOB), beat-cross frequency (BCF), amplitude of lateral head displacement (ALH), and dancing mean (DNM) of sperm showed significant positive or negative interrelationships among each other in both fresh (r = 0.41 to 0.91) as well as post-thawed (r = 0.44 to 0.90) semen. Moreover, the correlations of motility and kinematics parameters of total motile sperm in both fresh and frozen-thawed semen were highly significant with velocity/kinematics traits of only progressively motile sperm, and the velocity traits among only motile sperm were highly significantly interrelated in both fresh (r = 0.46 to 0.98) and frozen-thawed (r = 0.43 to 0.93) semen of Girbulls, though the magnitudes of correlations were lower in frozen-thawed semen as compared to fresh semen. Thus, CASA analysis offresh semen for motility and velocity traits could predict the post-thawed sperm motility and velocity/kinematics of bovine semen.

ObjectiveThe frozen semen has the significant advantages of long-term storage and long-range transportation. However, due to the low cryotolerance of boar sperm, the global utilization of frozen boar semen in artificial insemination was less than 1% until the year 2000.MethodsIn this study, the effects of five cryoprotectants at different concentrations on the cryotolerance of boar semen were evaluated when they were added separately, and the optimal concentrations for each cryoprotectant were determined, then their combined additive effects were further assessed.ResultsAt a glycerol (GLY) concentration of 5%, the quality of frozen-thawed sperm reached its maximum value, which was significantly higher than the 4% GLY group (p<0.05) and 0% GLY group (p<0.01). The straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), sperm plasma membrane integrity (SPMI), sperm acrosome integrity (SAI) and sperm mitochondrial activity (SMA) of the frozen-thawed sperm in treated egg yolk group exhibited significant improvements compared to the untreated egg yolk group (p<0.05). The total motility (TM), progressive motility (PM), SPMI, SAI, and SMA of 2% Equex STM paste group were significantly higher than the rest groups (p<0.05). The TM, PM, VSL, VCL, and VAP of frozen-thawed sperm in the 250 nM and 300 nM Mitoquinone mesylate groups showed significant improvements compared to the other groups (p<0.05), and the reactive oxygen species levels in sperm cells were also significantly lower (p<0.05). The quality of frozen-thawed boar sperm in 0.6 mM L-ergothioneine group reached its peak value and was significantly higher than the rest groups (p<0.05). When these five cryoprotectants were used in combination, the quality of frozen-thawed boar sperm exhibited a significant improvement compared to when they were used individually (p<0.05). Utilizing the frozen-thawed boar semen to inseminate estrus sows, the reproductive performance of the sows did not differ significantly from the sows inseminated with fresh semen (p>0.05).ConclusionThe optimized boar semen cryopreservation system can substantially enhance the quality of frozen-thawed boar sperm, making it suitable for artificial insemination in pig farm.

The pattern of sperm transport and survival in the mare’s reproductive tract is different between fresh and frozen-thawed semen. A probable reason for this difference is the biophysiological changes in sperm during cryopreservation of equine semen. These changes can impair motility of stallion sperm after thawing. The aim of the present work was to test the effect of different caffeine concentrations on stallion sperm motility after thawing. One ejaculate of 9 stallions was frozen with the INRA82 frozen extender, and after thawing, different caffeine concentrations were added to the semen samples according to the treatments: control INRA82 without caffeine addition (T1), T1+1mM caffeine (T2), T1+2mM caffeine (T3), T1+3mM caffeine (T4), T1+5mM caffeine (T5), T1+7.5mM caffeine (T6), and T1+10mM caffeine (T7). The analysis of sperm motility parameters was performed with a computer-assisted semen analyser in 4 time periods: immediately after semen samples thawing (t0) and 15min (t15), 30min (t30), and 40min (t40) after semen sample thawing. One semen sample of each treatment was thawed, and an aliquot was analysed for the following computer-assisted semen analysis characteristics: velocity curvilinear (VCL; µm s−1), velocity straight line (µm s−1), velocity average path (µm s−1), linearity (%), straightness (%), wobble (%), amplitude of lateral head displacement (µm), beat cross frequency (BCF; Hz), and percentage of total sperm motility (TM) and progressive sperm motility. The statistical analysis was performed with ANOVA and Duncan’s test. The sperm parameters progressive sperm motility, linearity, wobble, and amplitude of lateral head displacement did not differ among the treatments (P&amp;gt;0.05). Immediately after addition (t0) of 5, 7.5, and 10mM caffeine concentrations, an increase of TM was observed (T5: 53.1%; T6: 45.9%; and T7: 47.4%) compared with the other treatments (T1: 37.5%; T2: 36.0%; T3: 36.6%; and T4: 32.3%; P&amp;lt;0.05). Although after 15min of incubation (t15) the TM decreased compared with t0 in T5, T6, and T7 treatments, the percentage was comparable with the other treatments at t15, t30, and t40. The mean value for TM was higher with 5mM caffeine compared with the control group (38.6% v. 34.7%; P&amp;lt;0.05), whereas for the 10mM caffeine treatment velocity straight line (19.9v. 17.1µm s−1), velocity average path (25.6v. 22.9µm s−1), and straightness (75.4v. 72.3%) were higher than the control (P&amp;lt;0.05). For the 5, 7.5, and 10mM caffeine treatments, VCL and BCF were higher than the control (VCL: 33.9, 34.5, 36.8, and 31.5µm s−1, respectively; BCF: 8.1, 8.6, 9.0, and 7.2Hz, respectively). The remaining motility parameters did not differ until 40min after the treatment (P&amp;lt;0.05). In conclusion, the addition of 5, 7.5, and 10mM caffeine concentrations after semen thawing increased TM and most of the sperm motility characteristics. However, given the complexities of sperm transport, capacitation, and so on, further experiments are needed to test whether caffeine treatments could be used to improve the fertilization rate of frozen-thawed equine semen.

The present study investigated the relationship between seminal parameters and bull conception rate (BCR) in 72 Murrah bulls using Computer-Assisted Sperm Analysis (CASA). The BCR was calculated by obtaining data of artificial insemination spanned over two decades from buffalo farms of two organised herds of India. The association of seminal parameters and BCR was studied using multiple regression and principal component analysis. The average BCR was 38.95% ± 1.51%, ranging from 22.50% to 80.51%. Total motility (TM), progressive motility (PM), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head displacement (ALH) showed significant positive correlations with BCR, whereas straightness (STR) and linearity (LIN) were negatively correlated. Multiple regression (R2 = 0.29) identified TM as the most reliable predictor of BCR. Principal component analysis (PCA) extracted three components, PC1 (Sperm velocity and head movement, 54.36% variance), PC2 (Trajectory and beat frequency, 19.56%), and PC3 (Progressive motility and path accuracy, 12.98%), explaining 86.90% of the total variance. Regression using PC scores (R2 = 0.24) indicated positive effects of PC1 and PC3, and a negative effect of PC2 on fertility. Overall, sperm velocity and progressive motility were primary fertility determinants, while excessive linearity hindered conception success. Therefore, association of seminal parameters with BCR can be explored for enhancing breeding efficiency of bulls. Future breeding programmes should prioritise sperm velocity and progressive motility traits while avoiding excessive linearity to improve bull fertility and conception success.

We previously reported that single layer centrifugation (SLC) with Percoll® (GE Healthcare, Uppsala, Sweden) of fresh bovine semen resulted in improved sperm progressive motility and movement, as evidenced by computer-assisted sperm analyzer (CASA) after freezing-thawing. However, no report has been found in the literature on the use of Percoll Plus® (PP; GE Healthcare), a nontoxic colloid, for the same purpose. Thus, this study aimed to verify the effects of SLC-PP before bull sperm freezing on sperm kinematics after cryopreservation. Ejaculates were collected from 3 Nellore bulls (6 from each) using an artificial vagina. After collection, the semen was assessed and pooled, and then 1 billion spermatozoa either diluted [D; 1:2 (v/v)] in freezing extender (FE, without glycerol) or undiluted (UD) was layered on top of a 9-mL column of PP (in 15-mL centrifuge tubes) at concentrations of 70% or 90% to form the 70D, 70UD, 90D, and 90UD treatment groups. Following centrifugation for 13 min at 839 × g [except for the control (C) group], the supernatant was removed and the sperm pellet diluted to 50 × 106 sperm mL−1 in FE medium plus glycerol. Then, frozen–thawed sperm samples were analysed by CASA (MMC Sperm, St. Petersburg, Russia) for the following parameters: total motility (TM, %), progressive motility (PM, %), curvilinear velocity (VCL, µm−1), straight line velocity (VSL, µm s−1), average path velocity (VAP, µm s−1), amplitude of lateral head displacement (ALH, µm), beat cross frequency (BCF, Hz), linearity (LIN, %), and straightness (STR, %). For statistical analyses, ANOVA and Student-Newman-Keuls test were used. Data are presented as mean ± SEM with P &lt; 0.05 taken as significant. No difference was found among the groups for TM, VSL, BCF, and STR. However, the percentage of PM was higher (P &lt; 0.05) in the SLC-selected sperm samples (values ranging from 42.0 ± 7.0 to 47.4 ± 11.4) than in C (28.8 ± 5.0), and ALH was lower in 70UD (1.6 ± 0.12) and 70D (1.7 ± 0.10) than in C (1.9 ± 0.2). Moreover, 70UD (49.0 ± 1.0), 90UD (50.0 ± 3.0), and 90D (50.0 ± 4.0) displayed higher percentage of LIN (P &lt; 0.05) compared with C (45.0 ± 2.0) and 70D (48.0 ± 3.0). On the other hand, similar results were obtained for VCL (from 126.3 ± 8.0 to 130.0 ± 20.5) and VAP (from 82.7 ± 14.5 to 85.1 ± 6.9) in C, 70UD, and 70D, but these values differed (P &lt; 0.05) from those for VCL in 90UD (104.6 ± 10.3) and 90D (97.2 ± 22.0) as well as for VAP in 90UD (72.2 ± 11.0) and 90D (71.8 ± 9.6). These are the first data demonstrating favourable influences of SLC with 70% Percoll Plus® to select distinct sperm subpopulations as evidenced by enhanced PM, LIN, and ALH. Thus, SLC-PP could optimize the production of frozen bull semen by decreasing the number of sperm per insemination dose, and help to circumvent limitations associated with the poor semen quality sometimes found in bulls of high genetic merit. This research was funded by FAPESP # 2015/20986-3, MasterFertility and Tairana Artificial Insemination Station, Brazil.

Fertility response of artificial insemination methods in sheep with fresh and frozen-thawed semen

This study aimed to investigate the effects of refrigeration and cryopreservation on the sperm kinetic and morpho-functional parameters of 5/8 Girolando bulls. Eleven ejaculates from each bull were collected, diluted, and divided into two portions: one portion was subjected to refrigeration at 5ºC for 24 and 48 hours, while the second portion underwent cryopreservation(-196ºC). Post-thawing, various kinetic parameters were analyzed using the Computer Assisted Sperm Analysis (CASA) system. These parameters included total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness coefficient (STR), wobble coefficient (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Additionally, plasma membrane integrity (PMI), acrosomal membrane integrity (AMI), mitochondrial membrane potential (MMP), and chromatin condensation (CC) were examined for morpho-functional analyses. The results indicated that kinetic parameters (TM, PM, VCL, VSL, VAP) and morpho-functional analyses (PMI, MMP) differed significantly (P &lt; 0.05) between cryopreserved and refrigerated semen, as well as other parameters such as LIN, ALH, and BCF across all groups. The acrosomal membrane integrity showed no differences, but CC varied significantly among all groups. In conclusion, semen from 5/8 Girolando bulls can be preserved for up to 48 hours at 5ºC, providing an alternative for the short-term use of genetic material from this breed.

Computer-assisted sperm class analyser (CASA) analysis of avian semen following cryopreservation indicates that their semen motility and viability parameters become compromised, due in part to oxidative stress. To mimic these observations we have treated cockerel semen with an oxidative stress inducing agent, namely hydrogen peroxide (H2O2) and monitored the motility, kinematic and viability parameters over time. Briefly, five healthy and fertile South African Venda cockerels were selected and their semen was collected using the abdominal massage technique. The semen was then treated with H2O2 at 0 &microM, 5 &microM, 50 &microM and 200 &microM concentrations for 0, 3, 16 and 24 hrs. The semen motility, kinematic and viability parameters were then determined using the CASA system while the viability was determined using the SYBR-14/PI staining. The Pearson's correlation coefficient was determined to test the relationships between the levels of induced oxidative stress, period of exposure to oxidative stress inducing agent and the motility plus kinematic parameters. Our data revealed that in raw cockerel semen, there was high and positive correlations between total motility (TM), progressive motility (PM), rapid (RAP), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) while the kinematic parameters LIN, STR, WOB, ALH and BCF had low or negative correlations with them. Furthermore, TM, PM, RAP, VCL and VSL remained highly and positively correlated with the induced oxidative stress and also, linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) remained negatively correlated with the induced oxidative stress, after 3 hrs. After 24 hrs, TM, PM, RAP, VCL, VSL, VAP and ALH, became negatively correlated with the induced oxidative stress while LIN, STR, WOB and BCF became positively correlated with the induced oxidative stress. Conversely, when the H2O2 concentration used was correlated with motility and kinematic parameters over time, TM, PM, RAP, VCL, VSL, VAP became negatively correlated with oxidative stress while LIN, STR, WOB, ALH and BCF show negative or low correlations with the induced oxidative stress. This data indicates that LIN, STR, WOB, BCF and to some extend ALH, reveal the least correlations with the induced oxidative stress under persistent oxidative stress conditions in cockerel semen. In conclusion, cockerel semen, like buck semen, does not easily succumb to oxidative stress since the raw semen correlations of CASA analysed parameters are comparable to these observed after 3 hrs of H2O2 treatment. In addition, the oxidative stress levels tolerated by cockerel semen should not 5 &microM H2O2 oxidative stress levels. Lastly, lack of correlation between LIN, STR, WOB, BCF and ALH and induced oxidative stress can be used in cockerel semen to show intolerable cryopreservation conditions.

Sperm Motion Characteristics May Discriminate Fertile From Infertile Men With Normal Parameters

Effects from aging on semen quality of fresh and cryopreserved semen in Labrador Retrievers

Quantification of damage at different stages of cryopreservation of endangered North American bison ( Bison bison) semen and the effects of extender and freeze rate on post-thaw sperm quality