Removal Of Seminal Plasma Research Articles

Seminal plasma removal for medium-term preservation of ram sperm at 5 °C

This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

Proteomic profile of seminal plasma from Pêga donkeys (Equus asinus) with high sperm motility and vigor: Implications for assisted reproduction

Donkeys of the Pêga breed (Equus asinus) have been used for two centuries to produce breeding stock and create hybrids for labor and transport in southeast Brazil, and for exporting meat and milk to other countries. Furthermore, they are used in competitions, as they are docile and easy to handle. However, assisted reproduction success rates for frozen donkey semen are remarkably low, with no standardized method for cryopreserving sperm after removal of seminal plasma. This work aims to reveal the biological involvement of seminal plasma proteins from Pêga donkeys in aiding the development of assisted reproduction. This study was carried out with 14 ejaculates collected every eight days, throughout the breeding season, from three healthy fertile Pêga donkeys, with an average age of four years. After confirming the high freezability of fresh semen by evaluating quality parameters, the seminal plasma was separated by centrifugation and an aliquot from each collection was microfiltered and frozen. A label-free technique followed by LC-MS/MS analysis applied to pools of seminal plasma samples from each animal revealed 522 proteins in the proteomic profile, of which 49.8 % (260 proteins) are related to cellular energy transformation, and many proteins involved in reproduction (76), spermatogenesis (38), fertilization (29), among other biological process. By comparison with literature, Pêga donkeys share many proteins with donkeys of Dezhou breed that present great potential as fertility biomarkers. Our results showed proteins positively related to fertilization for different breeds of donkeys around the world, helping to enhance the assisted reproduction of Pêga donkeys.

Freezing Stallion Semen-What Do We Need to Focus on for the Future?

Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation.

Qualitative indicators of sperm from producing goats before and after cryopreservation

Currently, artificial insemination, as one of the types of assisted reproductive technologies, is widely used in dairy and beef cattle breeding. The same cannot be said about such a promising direction in the agricultural industry as goat breeding. One of the limiting factors is the negative impact of low temperature on the morphofunctional charateristics of sperm of breeding goats. The purpose of this research was to test a protocol of sperm cryopreservation of stud goats with modification of prepreparation and subsequent assessment of the quality indicators of sperm before and after its deep freezing. A comprehensive assessment of sperm quality (volume, concentration, morphology, motility) of goats (n=10) was carried out using generally accepted methods and protocols. The assessment of sperm quality indicators included five stages: after sperm collection, two hours after cooling, after thawing: 0 hours, 1 and 2 hours. According on the obtained results, the sperm of breeding goats ha low cryoresistance. After cryopreservation (0, 1 and 2 hours after thawing), there is an increase in the number of sperm with tail damage by 7.5% (p≤0.05), 15.5% and 21.8% (p≤0.01), and also a decrease in the number of progressively moving sperm by 1.4; 1.6 and 2.5 times (p≤0.01) compared with the results of the assessment 0 hours after collection. The use of a deep two-phase sperm freezing protocol allows maintaining the viability of sperm with a progression of movements equal to 54.2±5.1% and a number of morphologically normal sperm equal to 64.1±1.9%. In this case, the prepreparation of sperm for the cryopreservation process (current protocol) includes sperm centrifugation (mode: 7000 rpm for 15 minutes), removal of seminal plasma, dilution 1:4 (OptiXcell diluent), cooling (4 hours at 4℃); sperm cryopreservation protocol: 1. immersion of goblets with paillettes 4 cm above liquid nitrogen for 7 minutes; 2. complete immersion in liquid nitrogen.

Sperm replacement in sperm-storage tubules causes last-male sperm precedence in chickens

ABSTRACT 1. This study elucidated the last-male sperm precedence (LMSP) mechanism in chickens by examining replacement in storage tubules (SSTs) after multiple artificial inseminations (AI) and the effects of seminal plasma (SP) and male breed on sperm replacement in SSTs. 2. Hens were artificially inseminated with fluorescent dye-labelled spermatozoa from White Leghorn (WL) chickens. Secondary AI was conducted 3 d later with sperm labelled with different nuclear fluorescent dye. Percentage of first and second inseminated sperm in SSTs and their logarithmic odds were calculated. The effect of SP on LMSP was examined using (1) Lake’s solution-washed sperm before second insemination, and (2) SP injected continuously after first insemination. Effect of breed difference on sperm replacement was investigated using Barred Plymouth Rock (BP) sperm. 3. Successive WL-sperm inseminations at three-day intervals caused > 70% stored sperm replacement in SSTs. Although SP removal from sperm from second insemination significantly decreased replacement, its intra-vaginal injection did not affect release. Secondary insemination using BP sperm significantly increased replacement. 4. Sperm replacement is a major factor favouring LMSP in domestic chickens. Two fluorescent staining of sperm, and intra-vaginal multiple AI technique have enabled visualisation, differentiation, and quantification of multiple inseminated sperm stored in the SSTs.

P-061 In situ microfluidics of fluidic walls: a novel deviceless and cost-effective approach for sperm selection in the same ICSI-dish

Abstract Study question Is our novel deviceless method based on in-situ microfluidics a valuable strategy to select suitable sperm for ICSI? Summary answer The novel protocol allows selecting sperm for ICSI within 15 minutes, in the same ICSI dish and disregarding fungible supplies, centrifugation and washing steps. What is known already Microfluidics is an innovative operation in ART which integrates sperm guidance biomimicry during the in vitro sperm selection process. In contrast to other sperm preparation methods such as Wash-Swim-Up and Density- Gradients-Centrifugation, microfluidics disregards washing and centrifugation steps along the procedure. In addition, microfluidics are time-efficient methods which reduce risks associated with handling, gamete mix-up and ROS production. However, microfluidics devices are costly and likely to fail to select motile sperm in dispermic samples. With the aim of outlining the application of microfluidics in ART, we conceived a lab-on-a-chip approach for sperm selection by in-situ handmade microfluidics of fluidic walls. Study design, size, duration The microfluidics circuit conforms to Laplace and hydrostatic pressure laws and integrates the sperm guidance mechanisms of rheotaxis and boundary-following behavior. System’s efficacy is based on the volume differences, the distances between microdoplets, and their arrangement. It divides into Section 1 (dispersion and seminal plasma removal) and Section 2 (sperm sorting in response to positive rheotaxis). The system was adjusted to recover at least 20 selected progressive sperm for ICSI in less than 15 minutes. Participants/materials, setting, methods We prepared the system on a conventional IVF polystyrene round dish (15 mm diameter) using MOPS buffer, PVP and mineral oil only. We verified the flow-driven dynamics by registering the horizontal displacement (direction) and flow velocity (µm/s) using eosin-Y and methylene blue dyes. We confirmed the removal of the seminal plasma in a PSA assay. The proof-of-concept analysis was performed using fresh semen samples from 150 patients (Caucasian; mean age: 33,53±8,19). Main results and the role of chance Our design includes three main points to allow sperm separation: 1 (initial), 2 (rebalancing), 3 (final) with a total distance of 1,5 cm between 1 and 3. Our system assures a continuous microfluidic flow, with two opposed directions (from point 1 to point 2 and from point 3 to point 2). PSA assay showed a significant decrease in PSA levels >200000 ng/ml in the fresh sample to undeterminable levels (<0,0091 ng/ml) at point 3. To assess the utility to select a suitable number of sperm for ICSI we used sperm samples with the following characteristics: volume 3,6 (1,0-7,6) ml, concentration 68 (0,29-150) ×106/ml, progressive motility 24 (2-41) % and morphology 6 (1-15) % normal. The minimum and maximum times for sperm recovery at point 3 were recorded at 5 and 35 minutes, respectively. Normozoospermic samples required lower recovery times compared to dispermic (p < 0,001), although according to the median, samples with at least 1 × 106/ml of motile sperm need less than ten minutes recovery time. Sperm parameters: concentration, motility and morphology were inversely correlated with the recovery time (p < 0,0001), being morphology the parameter with the lesser influence on the outcome. Limitations, reasons for caution The present protocol depends on a free-hand preparation. Although the use of templates and the training reduces inter and intra-operator variability, a ready-to-use surface would benefit the operability. Non-inferiority studies are required to compare our method with the currently used ones (Wash-Swim-Up or Density-Gradients-Centrifugation) before clinical implementation. Wider implications of the findings Our deviceless methodology streamlines sperm selection for ICSI and reduces risks along the procedure. This novel approach represents a paradigm shift in sperm preparation, since it dispenses with centrifugation, washing and plasticware. The present protocol can be implemented to the clinic in the near-term, particularly due to its simplicity. Trial registration number na

Different Methods for Seminal Plasma Removal and Sperm Selection on the Quality and Fertility of Collared Peccary Sperm.

Methods for seminal plasma (SP) removal and the selection of collared peccary sperm for fertilization were compared. The experiments evaluated the following: the (I) impact of centrifugation for SP removal before swim-up for sperm selection and (II) a comparison of different Percoll® gradient densities (PG 45-90% and PG 35-70%). Non-selected sperm served as the control. Sperm quality was assessed based on motility patterns, morphology, membrane functional integrity, viability, reactive oxygen species (ROS), glutathione (GSH), and DNA integrity. Subsequently, the most successful group in the previous experiment and washing by centrifugation (WC) were compared for motility patterns and fertilization using pig oocytes. Swim-up decreased motility and enhanced ROS compared to the control. Centrifugation before swim-up harmed integrity and viability compared to the control. PG 45-90% (96.8 vs. 69.7 vs. 40.7 µm/s) allowed for a better velocity average pathway (VAP), a better velocity straight line, and better linearity (LIN) than those of the control and PG 35-70% (88.4 vs. 56.0 vs. 27.3 µm/s). Thus, PG 45-90% was used for fertilization. PG 45-90% obtained a higher VAP, a higher amplitude of the lateral head, straightness, and higher LIN than those of the control and WC. Cleavage (25.2-26.3%) and morula (8.1-10.5%) rates did not differ between the groups. Therefore, PG 45-90% and WC were efficient in isolating collared peccary sperm capable of fertilizing pig oocytes.

Impact of seminal plasma on the equine endometrial transcriptome

Establishment of pregnancy involves a fine-tuned balance between protection and tolerance within the maternal immune system, as the mare accepts the semi-allogenic fetus while still being capable of combating uterine pathogens. The first uterine exposure to paternal antigens is during mating when the endometrium and draining lymphatics are introduced to semen. Seminal plasma (SP) plays an important role in preparing the female tract for a suitable immunologic environment in other species. The role of SP has been determined in the immediate inflammatory response to breeding, but its impact on the endometrium at the initial time of exposure to an embryo has not been investigated in the horse. Therefore, the objective of this study was to evaluate the endometrial transcriptome following insemination either with or without SP. We hypothesized that removal of SP would affect transcripts relating to immunotolerance, antigen presentation, and embryo growth and development. Six (n=6) mares were inseminated over four estrous cycles. Upon the detection of a pre-ovulatory follicle (>35mm), mares were assigned to one of three treatment groups: 1) uterine infusion with 30mL lactated Ringer's solution (LRS; negative control), 2) AI with 500 × 106 progressively motile sperm (PMS) in conjunction with 30mL SP (SP+), and 3) AI with 500 × 106 PMS without SP (reduced via gradient centrifugation), and resuspended in 30mL LRS (SP-). The order of SP+ and SP- wasrandomly assigned in a switch-back design, and separated by a washout cycle in which mares received a uterine infusion with 30mL LRS. hCG was administered to standardize the time to ovulation and endometrial biopsies were collected 7 days after insemination. RNA was isolated utilizing Trizol, and RNASeq was performed using Illumina NovaSeq6000, with 97.79% Total Mapping and 40 million read depth. The False Discovery Rate (FDR) was set to <0.05. In total 2100 genes were differentially expressed between all groups. When comparing the SP+ and SP- groups, 241 differentially expressed genes (DEGs) were identified. Biological processes that were impacted by the presence of SP included antigen processing and regulation, cholesterol synthesis, and immune/inflammatory response. Differentially expressed genes associated with these pathways included multiple genes relating to antigen presentation (HLA-DM Beta Chain, HLA-DRB, HLA-DQA, RASGRP1), immune cell signaling (CXCL9, CXCL1, B-defensin, MIP-2B), embryo growth and development (Activin, KLF2, RDH10, LAMA3, SLC34A2), and embryo metabolism (ABCA1, ABCA2, APOA1, LDL, INSR, IGFBP2, IGFBP3). In conclusion, the presence of SP had a significant effect on the endometrial transcriptome at the time when an embryo first is exposed to the uterine environment. Further research will evaluate the impact of these alterations on embryo maturation, placental development, and pregnancy outcome.

Assessment of methods to activate epididymal sperm motility

Horse epididymal sperm has satisfactory motility activation (Mot-Act) after harvesting and cryopreservation; however, donkeys typically display poor Mot-Act despite high-plasma-membrane integrity (PMI) with high-mitochondrial-membrane-potential (ΔΨM). Mot-Act can be achieved in murine epididymal sperm with centrifugation by removal of sperm-bound seminal plasma (SP) proteins; also, in other mammals, the dilution of epididymal sperm with SP promotes Mot-Act. Dilution of cryopreserved sperm with an extender without cryoprotectants aids Mot-Act in other species. It is possible that all these methods can enhance donkey and horse epididymal Mot-Act and that there might be positive interactions between methods; however, these hypotheses have yet to be tested. Single-layer colloid-centrifugation (SLC) is a powerful tool to remove sperm-bound particles. Thus, this study sought to assess methods and potential interactions to promote Mot-Act in epididymal sperm by comparing an equid with satisfactory Mot-Act (i.e., the horse) with another poor Mot-Act (i.e., the donkey). Mot-Act, PMI, and ΔΨM were assessed by submitting horse and donkey cryopreserved sperm to re-extension (1:3) on the same egg-yolk extender used for cryopreservation (BC), also the same extender lacking CPA, and donkey- and horse-SP. The Mot-Act, PMI, and ΔΨM were carried out 10-, 30-, and 60-min after incubation. Results showed that donkey and horse epididymal sperm had lower Mot-Act, PMI, and ΔΨM when dilutedwith donkey- and horse-SP (P<0.05). Conversely, Mot-Act was similar between horse-SP, BC, and CPA (P>0.05), and it all increased after 10 min of incubation (P<0.05). PMI and ΔΨM were not different across groups in horse epididymal sperm (P>0.05). Then the same processing methods were applied before and after SLC for cryopreserved horse and donkey epididymal sperm, and then Mot-Act, PMI, and ΔΨM were assessed. The results showed that donkey epididymal sperm had higher Mot-Act when diluted in the same egg-yolk extender compared to donkey-SP or CPA (P<0.05); Horse epididymal sperm had lower PMI and ΔΨM when diluted in donkey-SP compared to BC and horse-SP (P<0.05). The sperm recovery after SLC was similar between donkey and horse epididymal sperm (P>0.05). Next, zona-pellucida binding was carried out as an in vitro functional assessment of Mot-Act, as it is necessary for fertilization. Dilution with SP from either species' sperm did not enhance zona-pellucida binding (P>0.05) as it does for other mammals. Previously, it was suggested that donkey sperm does not bind to heterologous zona-pellucida (i.e., bovine); however, herein, it did bind similarly to horse sperm (P<0.05). This study is a foundation to expand the knowledge in equine sperm biology, showing that donkey epididymal sperm can have Mot-Act enhanced using re-extension with BC, and other standard methods cannot. Also, this is the first report to demonstrate that donkey sperm binds to heterologous zona-pellucida.

Effect of clarified tris egg yolk citrate diluter and seminal plasma removal on plasma membrane integrity in cryopreserved Surti buck semen

Effect of clarified tris egg yolk citrate diluter and seminal plasma removal on plasma membrane integrity in cryopreserved Surti buck semen

142 Methods to concentrate stallion semen for storage

It has been demonstrated that removal of seminal plasma and resuspension of the sperm pellet in various diluents will improve the post storage motility of spermatozoa from some stallions. This normally involves centrifugation. However, a sperm filtering device marketed by Botupharma (Phoenix, AZ) allows removal of seminal plasma without use of a centrifuge. This study was designed with the objective of comparing spermatozoa recovery rates using this device vs. centrifugation with and without a cushion. Thirteen ejaculates were collected from 3 Quarter Horse stallions. Before treatment aliquots were diluted with Dulbecco's modified PBS at a dilution rate of 1:1. For the 2 centrifugation treatments, 12 mL of the diluted semen was placed in a 15 mL conical bottom tube and centrifuged for at 1000g for 20 min with Red Cushion, (Botupharma), or at 400g for 10 mintues without a cushion. Aliquots for the third treatment were also diluted 1:1 and 20mL of the solution and poured into a SpermFilter (Botupharma) for separation. To determine % of sperm recovered, the concentration of aliquots before and after filtration or centrifugation was measured using a densimeter device (ARS, Chino, CA). Data were analyzed by one-way ANOVA with Tukey's test used for pairwise comparisons. Average % recovery rates were 69.4%, and 60.9% for aliquots centrifuged without and with a cushion as compared with 77.6% for the SpermFilter treatment. In this experiment the % recovery was lower (P < 0.02) for those aliquots which were centrifuged with Red Cushion compared with those separated with the SpermFilter. These results could differ if the same procedures were compared with a different technician. Furthermore, because of the diluent used following separation no attempt was made to compare motility following the separation procedures.

Donkey semen cryopreservation: Alternatives with permeable, non-permeable cryoprotectants and seminal plasma.

Cryopreservation of semen is an important technique to preserve genetic material. Yet, pregnancy rates in jennies after artificial insemination with frozen-thawed donkey semen are poor. This condition has been attributed to the impact of permeable cryoprotectants, that could cause high post-breeding endometritis. Removal of seminal plasma (SP) prior to semen freezing process is another contributing factor. SP is involved in a multitude of sperm functions and events preceding fertilization and has a mediating effect of sperm capacitation and postcoital uterine inflammatory response. The aim of this study was to evaluate different alternatives in donkey semen cryopreservation with permeable, non-permeable cryoprotectants, BSA and SP. Thirty ejaculates from 10 donkeys were cryopreserved with different combinations of dimethylformamide (DMF, 5%), sucrose (SUC, 200 mM) and homologous SP (10%): DMF (T1), DMF/SP (T2), SUC/BSA (T3), SUC/BSA/SP (T4), DMF/SUC/BSA (T5), DMF/SUC/BSA/SP (T6), DMF/BSA (T7) and DMF/BSA/SP (T8). After thawing, sperm motility and kinetics were assessed by computerized semen analysis. Sperm vitality (SV) was evaluated by fluorescence microscopy, functional membrane integrity (FMI) by the HOST test, abnormal morphology by eosin-nigrosin staining and sperm membrane stability by flow cytometry. For statistical analysis, sperm quality indexes (SQi) were obtained, general linear models were carried out and mean comparisons were made by the Tukey test. T1, T2, T5, T6, and T7 had higher and equivalent results for motility, most kinetic parameters and function membrane integrity. Cryopreservation of donkey semen without permeable cryoprotectant (T3 and T4) showed a reduction in motility, kinetics, SV, FMI and SQi. T5 showed a reduction in progressive motility, sperm velocities, IMF and SQi compared to other DMF treatments. T6 and T8 achieved higher SQi values compared to T1, but they were not different compared to T2 and T7. T1 had a smaller sperm population with low-M540 compared to T3. It is concluded that the use of permeable cryoprotectant is essential to achieve higher post-thaw quality of donkey semen. In addition, the combined use of BSA, SUC and/or PS may provide additional sperm protection compared to the individual use of DMF.

Effect of Centrifugation Regime on Cryopreservation of Beetal Buck Semen

Background: Goat semen has phospholipase A enzyme which makes removal of seminal plasma an important step in its preservation. This study was aimed to standardize a centrifugation regime for cryopreservation of Beetal buck semen. Methods: A total of 36 pooled semen ejaculates were used for the experiment where initially 27 pooled ejaculates were utilized to find out the best time period out of 5, 8 and 11 minutes for each g-force viz., 700 x g, 1100 x g and 1400 x g using 9 pooled ejaculates per g-force. The remaining 9 pooled ejaculates were employed to find out the best centrifugation regime amongst the best time period for each g-force (8 minutes for 700 x g, 8 minutes for 1100 x g and 5 minutes for 1400 x g). Result: The percentage of sperm motility, live sperm, intact acrosome and HOST-reacted sperm differed significantly (P less than 0.0001) between time periods at 700 x g, 1100 x g and 1400 x g. The sperm parameters were significantly (P less than 0.05) higher at 1400 x g for 5 minutes and 1100 x g for 8 minutes than at 700 x g for 8 minutes. In conclusion, adoption of a high centrifugation force for a short duration of time for washing could improve the quality of frozen spermatozoa.

Ability of donkey sperm to tolerate cooling: Effect of extender base and removal of seminal plasma on sperm parameters and fertility rates in mares.

Artificial insemination using cooled-transported semen has marked importance in equine breeding programs around the world, and the high value of mules has generated avid interest in donkey semen biotechnology. However, donkey semen cools poorly in commercially available equine extenders. Therefore, this study aimed to develop approaches to improve the ability of donkey semen to tolerate cooling. Ejaculates of seven donkeys (n = 21) were cooled at 5°C for 48 h in three different extenders (milk-based, SM; sodium caseinate-based, SC; or egg yolk-based, EY) in the presence or absence of seminal plasma (centrifugation, C). Sperm motility, plasma membrane integrity (PMI), plasma membrane stability (PMS), mitochondrial membrane potential (HMMP), intracellular hydrogen peroxide (H2O2), and intracellular superoxide () were assessed before, 24 h, and 48 h post-cooling. In addition, 15 mares (163 estrous cycles) were randomly inseminated with semen from two jacks (Jack 1, n = 90; Jack 2, n = 73) previously cooled for 24 h under one of the treatments (SM, SC, EY, SM-C, SC-C, or EY-C). Groups EY, SC-C, and EY-C (P < 0.05) demonstrated superior sperm analytical parameters to SM at 24 and 48 h. Centrifugation positively affected sperm analytical parameters in cooled donkey semen extended in SM and SC (P < 0.05). Mares bred with semen extended in SC (67%, 18/27), SC-C (89%, 24/27), EY (89%, 25/28), or EY-C (74%, 20/27) had significantly greater conception rates than mares bred with SM (33%, 9/27; P < 0.05). Mares bred with SM-C had intermediate conception rates (59%, 16/27). In conclusion, SC and EY improved the cooling ability and fertility of donkey semen in horse mares, and centrifugation positively affected donkey semen extended in SM.

The Combined Effect of Melatonin Implant and Removal of Buck Seminal Plasma on Cryopreservation During the Nonbreeding Season.

This study aimed to determine how melatonin (MT) and seminal plasma affected the freezability of buck sperm during the nonbreeding season. Semen was collected from eight bucks before (pre-MT) and after (post-MT) MT application in the nonbreeding season. Individual ejaculates were collected from the bucks, split into two equal groups according to the removal of seminal plasma (SP) (-) or nonremoval of SP (+). For washing, the groups of ejaculates were centrifuged, and the supernatant was separated, SP (-) and SP (+) ejaculates were diluted, then frozen. Semen samples were examined for sperm motility, plasma membrane integrity, defective acrosomes, DNA fragmentation, and mitochondrial membrane function at the native and post-thaw stages. When the general average post-thaw motility (p < 0.01), plasma membrane (p < 0.05), acrosome (p < 0.05), and DNA integrity rates (p < 0.05) and mitochondrial membrane potential (MMP) (p < 0.01) were evaluated, it was seen that MT administration caused a statistically significant improvement. The dramatic effect of nonremoval of seminal plasma on motility and plasma membrane integrity is more clearly observed in individual semen samples frozen in the pre-MT group (p < 0.05). Also, it was observed that removing seminal plasma in the post-MT group caused even milder post-thaw acrosome damage compared with the SP (+) group (p < 0.05). The effect of removing seminal plasma was not observed in terms of DNA integrity and MMP rates in pre- and post-MT groups. As a result, it was concluded that MT application and removal of seminal plasma in the nonbreeding season result in improvement in the freezability of buck semen.

The effects of green synthesized anionic cupric oxide nanoparticles on Zaraibi goat spermatozoa during cryopreservation with and without removal of seminal plasma

Sperm motility, normal morphology, viability, spermatozoa DNA damage, and lipid peroxidation are all affected by semen cryopreservation. The goal of this study was to see how effective cupric oxide nanoparticles (CuONPs) are as a cryo-extender additive on post-thawed sperm parameters. An artificial vagina was used to collect semen samples from five mature Zaraibi bucks (2–3 years). Ejaculates were pooled and separated into two fractions (A&B), a fraction (A) was left without being centrifuged and a fraction (B) was centrifuged to remove seminal plasma. Both fractions were diluted with tris egg yolk citrate extender (TECE) and then divided into five equal aliquots, each supplemented with (0, 10, 20, 40, and 60 ppm/ml) CuONPs. The findings revealed that removing seminal plasma before cryopreservation harms sperm parameters. Sperm motility, viability index, membrane integrity, biochemical antioxidant marker, DNA integrity, and MDA level improved after supplementation with CuONPs up to 60 ppm/ml, the most prominent significant positive effect was obtained with the highest dose (60 ppm/ml) without removal of the seminal plasm compared to control group. In conclusion: The presence of seminal plasma with a high concentration of CuONPs (up to 60 ppm/ml) may help to mitigate the negative effects of cryo-preservation.

Effects of Heterologous Bovine Seminal Plasma-Supplemented Egg Yolk-Based Extender on Cryosurvivability of Pantja Buck Semen.

The present study was conducted to observe the effects of removal of seminal plasma of Pantja buck semen and supplementation of bovine seminal plasma (BSP) in the extender before cryopreservation. In a preliminary experiment, different levels of BSP were supplemented (1, 3, 5, 7, and 9% v/v) in egg yolk (7.5% egg yolk)-tris (EYT) extender and used for cryopreservation of Pantja buck semen. Results in terms of motility, viability, plasma membrane integrity, acrosome integrity, and lipid peroxidation showed that 5% BSP was suitable for maintaining Pantja buck semen quality during cryopreservation. In the final experiment, pooled semen from four Pantja bucks was split into three aliquots (I, II, and III). Aliquot I was directly diluted in EYT extender and grouped as the control (C); aliquot II and III were washed separately with TALP solution and diluted as D1 (Washed semen with EYT extender) and D2 (Washed semen with EYT extender containing 5% BSP), respectively. Seminal attributes (sperm individual motility, viability, plasma membrane integrity, acrosome integrity, and total morphological abnormalities) were assessed at the postdilution, postequilibration, and post-thawing stages. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) concentration, and glutathione peroxidase (GSH-Px) activity were measured at post-thaw. Washed semen significantly improved (p < 0.05) seminal parameters at post-thaw compared with unwashed semen (control). A significant difference (p < 0.05) was observed in seminal attributes between freezing stages and between dilution groups. Significantly higher (p < 0.05) post-thaw sperm motility, viability, plasma membrane integrity, acrosome integrity, and GSH-Px activity, and significantly lower (p < 0.05) MDA concentration and extracellular release of enzymes (ALT, AST) were observed in group D2 compared with control and D1. The results of the present study demonstrated that cryopreservation of washed Pantja buck semen diluted with 5% BSP-supplemented EYT extender can improve post-thaw semen quality.

The Proteomic Modification of Buck Ejaculated Sperm Induced by the Cryopreservation Process.

Using two-dimensional electrophoresis along with mass spectroscopy, we have investigated how the cryopreservation process affected the protein profile of goat ejaculated sperm. In this study, five bucks were used for semen collection. After removal of seminal plasma, the Tris-based extender containing glycerol and egg yolk was used to freeze semen. The results indicated that the post-thaw sperm quality showed a significant reduction compared with fresh sperm. The numbers of protein spots acquired in fresh and post-thaw sperm were 2926 ± 57 and 3061 ± 81, respectively. Twenty-two different abundant proteins (DAPs) were identified between fresh sperm and frozen-thawed sperm (≥3.0-folds, p < 0.05). The abundances of 19 proteins were significantly higher in the fresh sperm than the post-thaw sperm. The results of the gene ontology annotation showed the primary location of the DAPs on sperm cytoskeleton, protein complex, cytoplasm, and mitochondria. In addition, these proteins were mainly involved in ion binding, small molecular metabolic processes, structure molecule activity, guanosine triphosphatase activity, oxidoreductase activity, and protein complex assembly. The interaction networks among these DAPs demonstrated that they may play roles in oxidoreductase activity, structure, acrosomal function, and motility of sperm. Collectively, the proteome of goat sperm was altered during the cryopreservation process, demonstrating that protein modification induced by cryopreservation may be associated with the reduced quality of goat sperm after thawing.

What is the relevance of seminal plasma from a functional and preservation perspective?

When preserving sperm in the liquid or cryopreserved state, seminal plasma (SP) components within ejaculates can alter fertilizing capacity of these gametes. Depending on the species or how semen is collected, volume and concentration of SP components varies considerably. The SP contains substances essential for maintenance of sperm viability and fertility; however, these components can be deleterious depending on quantity, or duration of time before there is removal of SP from sperm in semen processing. Substances that impair (e.g., BSP – bull; HSP-1 – stallion; Major seminal plasma protein PSPI - boar) or improve (e.g., spermadhesin PSP-I - boar) spermatozoa fertilizing capacity have been identified. Depending on individual males, species, and semen collection procedures, SP removal may be beneficial before preservation in the liquid or cryopreserved state. In some cases, SP that is removed can be added back to thawing extender with there being positive effects in thawed sperm and for sperm viability in the female reproductive tract. In this review article, there is a focus on different effects of SP in samples of cooled and cryopreserved semen from four domestic species (pigs, horses, cattle, and sheep) with there being emphasis on how SP modulates the function and morphology of sperm cells before, during, and after preservation in the refrigerated or cryopreserved state. The present review is part of the Festschrift in honor of Dr. Duane Garner who made major contributions to the area of focus in this manuscript as evidenced by the many times his research is cited in this manuscript.

Normal seminal plasma could preserve human spermatozoa against cryopreservation damages in Oligozoospermic patients

BackgroundCryopreservation of human spermatozoa has been identified as an efficient procedure to preserve fertility in men before any cancer therapy or surgical infertility treatment. Despite the benefits of the procedure, the deleterious effects of cryopreservation have been proven on sperm structure and function. This study aimed to evaluate seminal plasma effects on human sperm characteristics after cryopreservation, and compare the addition of normozoospermic and oligozoospermic seminal plasma in the prepared oligozoospermic samples. Semen samples were collected from fifty-five oligozoospermic men and the twenty fertile individuals who referred to the infertility center. At first, a semen analysis was carried out on each neat ejaculate, and then some were cryopreserved. The remainder of the semen was divided into two, one for seminal plasma removal and the other for sperm preparation. Then, the prepared spermatozoa were cryopreserved in three groups: one with, and another without the addition of oligozoospermic seminal plasma, and still another with the addition of normal seminal plasma. After thawing, sperm DNA integrity, viability, motility, and morphology were determined.ResultsThe percentages of all parameters were significantly lower after cryopreservation in all groups compared to the fresh sample. However, this reduction was lower in the oligozoospermic samples cryopreserved with normal seminal plasma.ConclusionThe results indicated that seminal plasma in oligozoospermic patients could not support sperm against cryo-injuries, an indication likely due to insufficient antioxidants and other protective components in oligozoospermic patients. However, normal seminal plasma could slightly preserve sperm characteristics after cryopreservation in oligozoospermic patients.