The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

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116 EFFECT OF INCUBATION OF BOVINE EPIDIDYMAL SPERMATOZOA IN SEMINAL PLASMA PRIOR TO CRYOPRESERVATION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab116 ◽

2007 ◽

Vol 19 (1) ◽

pp. 175

Author(s):

C. A. Guerrero ◽

J. A. Jenkins ◽

J. W. Lynn ◽

K. R. Bondioli ◽

R. A. Godke

Keyword(s):

Seminal Plasma ◽

Sperm Quality ◽

Egg Yolk ◽

Quality Parameters ◽

Water Bath ◽

Sperm Chromatin ◽

Mitochondrial Activity ◽

Dna Integrity ◽

Epididymal Sperm ◽

Acrosome Integrity

Studies examining the influence of seminal plasma on sperm function have shown both beneficial and detrimental effects. However, its effect on pre-frozen bovine epididymal sperm (BES) has not been documented. The objective of this study was to determine the effect of a 30-min incubation of BES in bovine seminal plasma (SEMP) prior to freezing on post-thaw sperm parameters. Paired testes were obtained from mature bulls (n = 10) at a local abattoir and transported to the laboratory (25–28°C) within 3–5 h postmortem. BES were harvested by multiple incisions from the caudae epididymides of each bull, pooled, and split into either Treatment A, 3 mL of egg yolk Tris-based medium (EYT) (No SEMP), or Treatment B, 3 mL of SEMP, each for 30 min in a 37°C water bath. SEMP was extracted from ejaculates of mature bulls (n = 6), and then pooled and stored at −20°C until used. Sperm from both treatments were re-suspended in EYT, adjusted to 70 × 106 sperm mL−1 and cooled to 4°C at 0.1°C min−1. Samples were diluted slowly over a 30-min period with 1:1 EYT medium containing 14% glycerol and loaded into 0.5-mL straws. Straws were frozen in liquid nitrogen vapors. For sperm analyses, straws were thawed in a water bath at 37°C for 40 s. Morphology was determined by staining with eosin-nigrosin. Viability and acrosome integrity were measured simultaneously with the combination of SYBR 14, propidium iodide, and PE-PNA. Mitochondrial activity was measured by the combination of MitoTracker Red and SYBR 14. DNA integrity was determined by acridine orange using the sperm chromatin structure assay (SCSA®; SCSA Diagnostics, Inc., Brookings, SD, USA). All assays were performed by multicolor flow cytometry. Differences between treatments were analyzed using one-way ANOVA (P < 0.05). In summary, all sperm quality parameters decreased significantly after thawing in both treatments (Table 1). A significantly higher overall and progressive motility post-thaw was achieved when sperm were incubated in SEMP prior to freezing. However, no difference was detected in sperm viability, acrosome integrity, mitochondrial activity, and DNA integrity between treatments. Also, SEMP reduced the quantity of distal droplets and broken tails post-thaw over that of no SEMP. Results indicate that incubation of BES in bovine seminal plasma prior to freezing improves post-thaw sperm quality. Table 1. Effect of bovine seminal plasma (SEMP) on quality parameters (mean ± SEM) of post-thaw bovine epididymal sperm

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Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro

Reproduction ◽

10.1530/rep-12-0311 ◽

2012 ◽

Vol 144 (6) ◽

pp. 687-697 ◽

Cited By ~ 13

Author(s):

J Beek ◽

H Nauwynck ◽

D Maes ◽

A Van Soom

Keyword(s):

Sperm Quality ◽

Membrane Integrity ◽

Inhibitory Effect ◽

Fertilization Rate ◽

Quality Parameters ◽

Fertilization In Vitro ◽

Cumulus Oophorus ◽

Sperm Penetration

In this study, we report for the first time on a possible contribution of metalloproteases in sperm passage through the cumulus matrix in pigs. The presence of 20 μM 1,10-phenanthroline (1,10-PHEN), inhibitor of zinc-dependent metalloproteases, strongly inhibited the degree of sperm penetration in cumulus-intact (CI), but not in cumulus-free (CF), porcine oocytes during IVF. The inhibitory effect of 1,10-PHEN was due to the chelation of metal ions as a non-chelating analog (1,7-PHEN) did not affect IVF rates. Furthermore, incubation with 1,10-PHEN did not affect sperm binding to the zona pellucida nor sperm motility, membrane integrity, or acrosomal status. These findings led to the assumption that 1,10-PHEN interacts with a sperm- or cumulus-derived metalloprotease. Metalloproteases are key players in physiological processes involving degradation or remodeling of extracellular matrix. In vivo, their proteolytic activity is regulated by tissue inhibitors of metalloproteases (TIMP1–TIMP4). We tested the effect of TIMP3 on fertilization parameters after porcine IVF. Similar to 1,10-PHEN, TIMP3 inhibited total fertilization rate of CI but not CF oocytes and did not influence sperm quality parameters. Although the inhibitory effect was stronger in CI oocytes, TIMP3 also reduced the degree of sperm penetration in CF oocytes, suggesting the involvement of a metalloprotease in a subsequent step during fertilization. In conclusion, our results indicate the involvement of TIMP3-sensitive, zinc-dependent metalloprotease activity in sperm passage through the cumulus oophorus in pigs. The results should provide the basis for further biochemical research toward the localization and identification of the metalloprotease involved.

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Optimization of Sperm Cryopreservation Protocol for Mediterranean Brown Trout: A Comparative Study of Non-Permeating Cryoprotectants and Thawing Rates In Vitro and In Vivo

Animals ◽

10.3390/ani9060304 ◽

2019 ◽

Vol 9 (6) ◽

pp. 304 ◽

Cited By ~ 1

Author(s):

Giusy Rusco ◽

Michele Di Iorio ◽

Pier Paolo Gibertoni ◽

Stefano Esposito ◽

Maurizio Penserini ◽

...

Keyword(s):

Brown Trout ◽

Semen Quality ◽

Sperm Quality ◽

Egg Yolk ◽

Dna Integrity ◽

Experimental Conditions ◽

C 30 ◽

Thawing Rate

The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C (p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions.

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Characteristics of high-quality Asian elephant (Elephas maximus) ejaculates and in vitro sperm quality after prolonged chilled storage and directional freezing

Reproduction Fertility and Development ◽

10.1071/rd12129 ◽

2013 ◽

Vol 25 (5) ◽

pp. 790 ◽

Cited By ~ 6

Author(s):

J. K. O'Brien ◽

K. J. Steinman ◽

G. A. Montano ◽

C. C. Love ◽

R. L. Saiers ◽

...

Keyword(s):

Sperm Quality ◽

Membrane Integrity ◽

Elephas Maximus ◽

Mitochondrial Activity ◽

Dna Integrity ◽

High Quality ◽

Chilled Storage ◽

Progressive Motility ◽

Directional Freezing

The in vitro quality of spermatozoa from one elephant (Elephas maximus) was examined after chilled storage and directional freezing (DF). High-quality, non-contaminated ejaculates (77.6 ± 6.0% progressive motility, 3.9 ± 1.5 µg creatinine mL–1 raw semen, 2.7 ± 0.6% detached heads) were cryopreserved after 0 (0hStor), 12 (12hStor) and 24 h (24hStor) of chilled storage. At 0 h and 6 h post-thawing, total motility, plasma membrane integrity, acrosome integrity, mitochondrial activity and normal morphology were similar (P > 0.05) across treatments. In contrast, progressive motility, rapid velocity and several kinematic parameters were lower (P < 0.05) for 24Stor compared with 0hStor at 0 h post-thaw. By 6 h post-thaw, amplitude of lateral head displacement and velocity parameters (average pathway, straight-line and curvilinear velocity) were lower (P < 0.05) for 24hStor compared with 0hStor and 12hStor. DNA integrity was high and remained unchanged (P > 0.05) across all groups and processing stages (1.6 ± 0.6% of cells contained fragmented DNA). Results indicate that DF after up to 12 h of chilled storage results in a post-thaw sperm population of acceptable quality for artificial insemination. These findings have implications for the cryopreservation of sex-sorted spermatozoa, which typically undergo more than 12 h of chilled storage prior to sorting and preservation.

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Seminal Plasma: Relevant for Fertility?

International Journal of Molecular Sciences ◽

10.3390/ijms22094368 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4368

Author(s):

Heriberto Rodriguez-Martinez ◽

Emilio A. Martinez ◽

Juan J. Calvete ◽

Fernando J. Peña Vega ◽

Jordi Roca

Keyword(s):

Seminal Plasma ◽

Current Knowledge ◽

Inorganic Ions ◽

Vitro Fertilization ◽

Dna And Rna ◽

Model Animal ◽

Sexual Glands ◽

Species Specific

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA—the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

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Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze - thaw damage

Reproduction Fertility and Development ◽

10.1071/rd08238 ◽

2009 ◽

Vol 21 (4) ◽

pp. 571 ◽

Cited By ~ 23

Author(s):

T. Leahy ◽

J. I. Marti ◽

G. Evans ◽

W. M. C. Maxwell

Keyword(s):

Seminal Plasma ◽

High Speed ◽

Sperm Quality ◽

Membrane Integrity ◽

Weight Fraction ◽

Protein Supplementation ◽

Freezing Process ◽

Spermatozoa Motility ◽

Functional Integrity

Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n = four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37°C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze–thawing.

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Finding an Effective Freezing Protocol for Turkey Semen: Benefits of Ficoll as Non-Permeant Cryoprotectant and 1:4 as Dilution Rate

Animals ◽

10.3390/ani10030421 ◽

2020 ◽

Vol 10 (3) ◽

pp. 421 ◽

Cited By ~ 1

Author(s):

Michele Di Iorio ◽

Giusy Rusco ◽

Roberta Iampietro ◽

Maria Antonietta Colonna ◽

Luisa Zaniboni ◽

...

Keyword(s):

Dilution Rate ◽

Sperm Quality ◽

Membrane Integrity ◽

Quality Parameters ◽

Dna Integrity ◽

Osmotic Resistance ◽

Progressive Motility ◽

Nitrogen Vapor ◽

Combined Use ◽

Cryopreservation Protocol

The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 (p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.

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Validation of the Turkey Semen Cryopreservation by Evaluating the Effect of Two Diluents and the Inseminating Doses

Animals ◽

10.3390/ani10081329 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1329

Author(s):

Michele Di Iorio ◽

Giusy Rusco ◽

Roberta Iampietro ◽

Lucia Maiuro ◽

Achille Schiavone ◽

...

Keyword(s):

Sperm Quality ◽

Sperm Concentration ◽

Membrane Integrity ◽

In Vivo Test ◽

Sperm Analysis ◽

Fertilizing Ability ◽

Vitro Analysis ◽

Hatching Rates

This study was designed to test the fertilizing ability of cryopreserved turkey semen, and here, two experiments were performed: an in vitro analysis to assess the effects of Tselutin and Lake diluents and an in vivo test to determine the fertility and hatching rates by also studying the feat of three insemination doses (250, 400 and 600 × 106 sperm/hen). Pooled semen samples were diluted with Tselutin or Lake extender which contained 20% of dimethylsulfoxide and 1 mM of Ficoll at final sperm concentration of 3 × 109 sperm/mL. Thereafter, semen was packaged into straws and frozen on liquid nitrogen. The post-thaw sperm quality was evaluated considering motility (computer-aided sperm analysis—CASA system) and membrane integrity (flow cytometry). Significantly higher values of progressive motility and some kinetic parameters in semen frozen with Lake were found. When we compared the extenders in vivo, no significant effects were detected, whilst sperm concentration significantly affected both fertility and hatching rates, with the best results obtained with the sperm concentration of 400 × 106 sperm/hen. From the results obtained, it emerged that the extender type only affected sperm motility characteristics, not the fertilizing ability of frozen-thawed semen, while inseminating dose markedly affected fertility and hatching rates.

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Flow cytometric and microscopic evaluation of post-thawed ram semen cryopreserved in chemically defined home-made or commercial extenders

Animal Production Science ◽

10.1071/an13215 ◽

2015 ◽

Vol 55 (4) ◽

pp. 551 ◽

Cited By ~ 16

Author(s):

M. Emamverdi ◽

M. Zhandi ◽

A. Zare Shahneh ◽

M. Sharafi ◽

A. Akhlaghi ◽

...

Keyword(s):

Plasma Membrane ◽

Soybean Lecithin ◽

Sperm Quality ◽

Egg Yolk ◽

Mitochondrial Activity ◽

Flow Cytometric ◽

Microscopic Evaluation ◽

Open Question ◽

Ram Sperm

The present study was designed to determine the effect of three different extenders on ram sperm quality during a freeze–thawing procedure using flow cytometric and microscopic evaluations. Several in vitro qualitative analyses of post-thawed sperm parameters including motility and velocity parameters, plasma membrane functionality, total abnormality, capacitation status, acrosome integrity, mitochondrial activity and apoptosis features were considered. In the breeding season, seven ejaculates from each Zandi ram were collected routinely twice a week. Following semen collection, samples were pooled and equally divided into three aliquots. Each aliquot was diluted and frozen with one of the following extenders: (1) Tris-based extender containing 1.5% (w/v) soybean lecithin (TSL), as a chemically defined extender, (2) Bioxcell, a commercial soybean lecithin-based extender, and (3) Tris-based extender containing 20% (v/v) egg yolk (TEY). The results of the present study indicated no differences in total [TSL (55.8 ± 2.02%) vs TEY (50.2 ± 2.02%; P < 0.05)] and progressive motility of spermatozoa [TSL (26.2 ± 1.36%) vs Bioxcell (22.4 ± 1.36%; P < 0.05)]. Semen freezing by means of TSL resulted in a higher percentage of live spermatozoa (39.42 ± 1.81%) compared with TEY (29.17 ± 1.81%; P < 0.05), and a higher percentage of functional plasma membrane (50.8 ± 192%) compared with TEY (44 ± 1.92%) and Bioxcell (38.8 ± 1.92%; P < 0.05). The effect of extenders on sperm capacitation status showed that the percentage of post-thawed capacitated spermatozoa was higher in TEY (61.9 ± 1.48%) compared with that in TSL (56.6 ± 1.48%; P < 0.05). The evaluation of post-thawed spermatozoa indicated that the percentage of live spermatozoa with active mitochondria was higher in TSL (53.05 ± 2.31%) compared with Bioxcell (45.92 ± 2.31; P < 0.05) and the percentage of intact acrosome spermatozoa was higher in TSL (84.55 ± 2.51%) compared with TEY (74.91 ± 2.51%; P < 0.05). The use of TSL and Bioxcell extenders reduced the percentage of apoptotic spermatozoa (40.82 ± 2.07% and 42.22 ± 2.07%, respectively), compared with TEY (51.34 ± 2.07%; P < 0.05). Post-thawing dead spermatozoa were increased when semen was frozen by Bioxcell (25.69 ± 1.28%). The results of this study showed that TSL extender may provide stabile milieu and conditions for ram sperm cryopreservation compared with Bioxcell and TEY extenders. Whether TSL extender can improve the artificial insemination results remains, however, an open question.

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Manganese Provides Antioxidant Protection for Sperm Cryopreservation that May Offer New Consideration for Clinical Fertility

Oxidative Medicine and Cellular Longevity ◽

10.4161/oxim.2.3.8804 ◽

2009 ◽

Vol 2 (3) ◽

pp. 152-159 ◽

Cited By ~ 24

Author(s):

Ranjna S. Cheema ◽

Amrit K. Bansal ◽

Gurmail Singh Bilaspuri

Keyword(s):

Protective Effect ◽

Semen Quality ◽

Maximum Level ◽

Egg Yolk ◽

Quality Parameters ◽

Vitro Fertilization ◽

Bull Semen ◽

Protein Leakage ◽

A Chain

Reactive oxygen species (ROS) are generated by sperm metabolism. While, ROS are required for maturation, capacitation and acrosome reaction, they also modify many peroxidable cellular compounds. There is production of ROS during cryopreservation and frozen spermatozoa are highly sensitive to lipid peroxidation (LPO). Antioxidants exert a protective effect on the plasma membrane of frozen bovine sperm preserving both metabolic activity and cellular viability. Manganese (Mn++) is proved to be a chain breaking antioxidant in biological system. Therefore, we examined the role of (Mn++) during cryopreservation of cattle bull semen. Semen was divided into four parts and cryopreserved in egg-yolk-citrate extender + glycerol (EYC-G), EYC-G + 100 µM of Mn++, EYC-G + 150 µM of Mn++and EYC-G + 200 µM of Mn++. After four hours of cooling and 24 hrs of freezing, the spermatozoa were examined for percentage motility, Hypo-osmotic swelling (HOS), LPO and protein leakage. Addition of manganese to the semen during cryopreservation showed a protective effect and accounted for an increase in semen quality parameters [percentage motility, HOS percent and decrease in malondialdehyde (MDA) production and protein leakage]. The effect of manganese on motility and HOS was non-significant (p < 0.05) in cooled spermatozoa but significant with 150 µM of Mn++in frozen-thawed spermatozoa. MDA production and protein leakage decreased to a significant and maximum level (p < 0.05) on addition of 200 µM of manganese. The addition of manganese to EYC-G dilutor will improve the quality/fertility of semen, which will result in improvement of in vitro fertilization and artificial insemination success rate.

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