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

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

Animals ◽

10.3390/ani11123452 ◽

2021 ◽

Vol 11 (12) ◽

pp. 3452

Author(s):

Uchechi Linda Ohaneje ◽

Uchebuchi Ike Osuagwuh ◽

Manuel Alvarez-Rodríguez ◽

Iván Yánez-Ortiz ◽

Abigail Tabarez ◽

...

Keyword(s):

Seminal Plasma ◽

Sperm Quality ◽

Membrane Integrity ◽

Egg Yolk ◽

Quality Parameters ◽

Mitochondrial Activity ◽

Dna Integrity ◽

Vitro Fertilization

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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Effective freezing rate for semen cryopreservation in endangered Mediterranean brown trout (Salmo trutta macrostigma) inhabiting the Biferno river (South Italy)

Zygote ◽

10.1017/s0967199415000647 ◽

2015 ◽

Vol 24 (5) ◽

pp. 668-675 ◽

Cited By ~ 5

Author(s):

Nicolaia Iaffaldano ◽

Michele Di Iorio ◽

Angelo Manchisi ◽

Stefano Esposito ◽

Pier Paolo Gibertoni

Keyword(s):

Liquid Nitrogen ◽

Brown Trout ◽

Salmo Trutta ◽

Semen Quality ◽

Egg Yolk ◽

Freezing Rate ◽

Frozen Semen ◽

Fresh Semen

SummaryThis study was designed to determine: (i) the in vitro effects of different freezing rates on post-thaw semen quality of Mediterranean brown trout (Salmo trutta macrostigma) from the Biferno river; and (ii) the in vivo fertilization and hatching percentage of freezing rate giving rise to the best post-thaw semen quality.Pooled semen samples were diluted 1:3 (v:v) in a freezing extender composed of 300 mM glucose, 10% egg yolk and 10% dimethyl sulfoxide (DMSO). The extended semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen surface (1, 5 or 10 cm) for 10 min to give three different freezing rates. Semen samples were thawed at 30°C for 10 s. The variables assessed after thawing were sperm motility, duration of motility and viability.Our results clearly indicate a significant effect of freezing rate on post-thaw semen quality. Semen frozen 5 cm above the liquid nitrogen surface showed the best quality after freezing/thawing. Based on these in vitro data, 2 groups of 200 eggs were fertilized with fresh semen or semen frozen 5 cm above the liquid nitrogen surface. Fertilization and hatching rates recorded for eggs fertilized with frozen semen were significantly lower (25.4% and 22.5%, respectively) than the ones obtained using fresh semen (87.8% and 75.5%, respectively). An effective freezing protocol will allow for the creation of a sperm cryobank to recover the original population of Mediterranean brown trout in the Biferno river.

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Effect of whole and clarified egg yolk based extenders on post-thaw semen quality in bulls.

10.31220/agrirxiv.2021.00050 ◽

2021 ◽

Author(s):

Prosper Kamusasa ◽

Eddington Gororo ◽

Fungayi Primrose Chatiza

Keyword(s):

Semen Quality ◽

Sperm Quality ◽

Egg Yolk ◽

Quality Parameters ◽

Normal Morphology ◽

Semen Cryopreservation ◽

Bull Semen ◽

Inclusion Level ◽

Whole Egg

Abstract This study was conducted to evaluate the comparative cryoprotective effects of whole egg yolk and clarified egg yolk on post thaw sperm quality parameters and to determine the optimum clarified egg yolk inclusion level (10-20%) in semen extenders for Mashona bull semen cryopreservation. It was shown that there was a significant decrease in sperm quality variables following cryopreservation. Semen quality increased with the concentration of clarified egg yolk, indicating a positive relationship between egg yolk LDL concentration and maintenance of in vitro sperm quality. The 20% clarified egg yolk (CEY20) extender treatment gave post-thaw motility, viability and normal morphology values which were comparable to the control (20% whole egg yolk, WEY20). The 10% clarified egg yolk concentration gave the least post-thaw quality values and the greatest proportion of defective spermatozoa. This experiment found no advantage of replacing whole egg yolk with up to 15% clarified egg yolk in Mashona bull semen cryopreservation. However, 20% clarified and 20% whole egg yolk performed similarly in the maintenance of post-thaw sperm motility, viability and normal morphology.

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A Simple and Efficient Semen Cryopreservation Method to Increase the Genetic Variability of Endangered Mediterranean Brown Trout Inhabiting Molise Rivers

Animals ◽

10.3390/ani10030403 ◽

2020 ◽

Vol 10 (3) ◽

pp. 403

Author(s):

Giusy Rusco ◽

Michele Di Iorio ◽

Roberta Iampietro ◽

Stefano Esposito ◽

Pier Paolo Gibertoni ◽

...

Keyword(s):

Liquid Nitrogen ◽

Brown Trout ◽

Sperm Concentration ◽

Mediterranean Area ◽

Fertilization Rate ◽

Semen Cryopreservation ◽

Nitrogen Vapor ◽

C 30

The aim of our study was to test the effectiveness of a simple semen cryopreservation procedure, developed for cultivated salmonid, on the wild salmonid of the Mediterranean area and to evaluate the effect of different thawing rates and sperm-to-egg ratios. The semen of five individual males was diluted into a final extender concentration of 0.15 M glucose and 7.5% methanol and loaded into 0.25 mL plastic straws, and a final sperm concentration of 3.0 × 109 sperm/mL was obtained. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above the liquid nitrogen level for 5 min. The semen was thawed at 40 °C/5 s or 10 °C/30 s. The sperm cryosurvival was evaluated by examining in vitro the sperm motility parameters using the CASA system, followed by fertilization trials in vivo, using three different sperm-to-egg ratios 6 × 105, 4.5 × 105 and 3 × 105:1. The applied cryopreservation procedure resulted in remarkably high (85.6%) post-thaw sperm total motility, when the semen was thawed at 40 °C/5 s, whilst the highest fertilization rate (53.1%) was recorded for a sperm-to-egg ratio of 4.5 × 105:1. According to these outcomes, the cryopreservation procedure that was tested turned out to be effective for the wild population of Mediterranean brown trout and practical for the creation of the first European semen cryobank foreseen as part of our “LIFE” Nat.Sal.Mo. project.

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15 EFFECT OF SEMEN THAW METHOD ON CONCEPTION RATE IN DAIRY HEIFER HERDS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab15 ◽

2005 ◽

Vol 17 (2) ◽

pp. 157 ◽

Cited By ~ 1

Author(s):

M. Kaproth ◽

H. Rycroft ◽

G. Gilbert ◽

G. Abdel-Azim ◽

B. Putnam ◽

...

Keyword(s):

Mixed Model ◽

Linear Mixed Model ◽

Semen Quality ◽

Sperm Quality ◽

Field Trials ◽

Conception Rate ◽

Warm Water ◽

Dairy Heifer

Semen processed with procedures permitting a flexible thaw method is used to breed millions of cows yearly. “Pocket thawing” is widely used as an alternative to warm-water thawing with such semen. To pocket thaw, a straw is retrieved from cryostorage, immediately wrapped in a folded paper towel, and moved to a thermally protected pocket for 2 to 3 min of thawing within the pocket before AI gun loading. Published field data are lacking for comparisons of such a thaw method with those for semen prepared to permit flexible-thawing. We measured the effect of warm-water or pocket thaw on conception rate in four dairy heifer herds using semen prepared with methods previously optimized for flexible-thawing success. Semen processing (Anderson S et al. 1994 J. Dairy Sci. 77, 2302–2307) includes two-step whole-milk extension, static vapor tank freezing (0.5-mL straws), and IMV Digitcool mechanical freezing (0.25-mL straws). It is unclear which specific processing steps permit flexible thawing. These procedures have been developed using breeding results from decades of field trials by professional inseminators using both pocket and warm-water thaw. Semen prepared from each of 12 sires produced equal straw units at 10 and 15 million total sperm per straw, in both 0.5- and 0.25-mL straw packages. Professional inseminators used each combination evenly over 16 months. Additional commercial semen (55% of total) from the same source was used. The thaw methods alternated weekly. Thaw effect on conception status, from 70 day non-return data for 11,215 services (67.6% conception rate), was estimated by a generalized linear mixed model. Neither thaw method nor total sperm per straw significantly affected conception rate (P = 0.658, 0.769, respectively). Bull, herd, inseminator within herd, year, season, and straw size did significantly affect conception rate (P < 0.05). No thaw method interactions with herd, sperm number, season, and straw package size were significant (P = 0.297, 0.526, 0.365, 0.723, respectively). This suggests that if semen has been prepared with procedures specific to flexible-thawing, it can be either pocket thawed or warm-water thawed within a range of herdsman or inseminator practices, season, or straw packaging choices. Even at 10 million, the lowest total sperm per straw, pocket thaw was equally as successful as warm-water thaw. We generally observe that in vitro sperm quality, as expected, is maximal for rapidly thawed straws, with slower thawing resulting in lower values. However, while it appears that conventional measures of in vitro semen quality are improved with fast thaw rates, these measures do not appear to correspond to higher in vivo fertility for semen prepared intentionally to be flexibly thawed. We conclude that, for semen prepared with procedures that permit flexible thawing, the thaw method, whether pocket or warm-water thaw, does not affect conception under commercial conditions and with routine semen handling methods. We thank the herd owners and their staff, the inseminators, and Hap Allen, Ron Hunt, Gordon Nickerson, and Bryan Krick of Genex for their help and cooperation.

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Making the most of sperm activation responses: experiments with boar spermatozoa and bicarbonate

Reproduction Fertility and Development ◽

10.1071/rd17476 ◽

2018 ◽

Vol 30 (6) ◽

pp. 842 ◽

Cited By ~ 4

Author(s):

William V. Holt ◽

Nana Satake

Keyword(s):

Semen Quality ◽

Current Knowledge ◽

Sperm Quality ◽

Environmental Chemicals ◽

Information Value ◽

In Vitro Tests ◽

Sperm Activation ◽

Activation Mechanisms

Attempting to extract useful and reliable information about semen quality and its fertility potential remains a difficult exercise, partly because the sperm heterogeneity within samples often renders simple statistical analyses rather meaningless. In fact, a mean and standard deviation may reflect neither the very fast swimming activities of the most active cells nor the slow and sluggish activities of others. Herein we propose that the information value within semen samples can be maximised if current knowledge about sperm activation mechanisms is exploited before undertaking the measurements. We explain, using boar semen as an example, that estimating and defining relative sperm subpopulation sizes, after activation by bicarbonate, provides a means of quantifying sperm quality. Although such estimates may indeed be related to in vivo fertility, the general approach also suggests potential new avenues that could be exploited for the elaboration of novel in vitro tests for the characterisation of toxic environmental chemicals and, indeed, to reduce the number of animals used in such testing programs.

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Effect of a pre-freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics

Reproduction Fertility and Development ◽

10.1071/rd12079 ◽

2013 ◽

Vol 25 (6) ◽

pp. 935 ◽

Cited By ~ 8

Author(s):

C. Tomás ◽

E. Blanch ◽

A. Fazeli ◽

E. Mocé

Keyword(s):

Dna Fragmentation ◽

Sperm Quality ◽

Epithelial Cell Line ◽

Dna Integrity ◽

Sperm Longevity ◽

Boar Sperm ◽

Fragmentation Dynamics ◽

Freezing Treatment

The aim of this work was to examine how a pre-freezing treatment with cholesterol-loaded cyclodextrins (CLC) affects boar sperm longevity, capacitation dynamics, ability to bind to a porcine telomerase-immortalised oviductal epithelial cell line (TERT-OPEC) in vitro and DNA integrity dynamics after freeze–thawing. Although the samples treated with CLC exhibited lower sperm quality than the control samples (P < 0.05) immediately after thawing, these differences disappeared (P > 0.05) after long-term incubation (26 h at 37 or 16°C). Additionally, the CLC-treated spermatozoa underwent similar capacitation and DNA fragmentation dynamics as the control spermatozoa (P > 0.05). However, CLC-treated spermatozoa were better able to bind to TERT-OPEC in vitro (P < 0.0001). In conclusion, the pre-freezing treatment of boar spermatozoa with CLC enhanced the ability of the spermatozoa to bind to TERT-OPEC in vitro, which could have an effect on the establishment of the sperm reservoir in the ampullary–isthmic junction in vivo. Additionally, frozen–thawed spermatozoa can be stored at 16°C for at least 6 h without a significant observable decline in sperm quality, which could be beneficial for the transport of thawed diluted doses of spermatozoa from the laboratory to the farm.

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Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism

Scientific Reports ◽

10.1038/srep31281 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 16

Author(s):

Richard G. Lea ◽

Andrew S. Byers ◽

Rebecca N. Sumner ◽

Stewart M. Rhind ◽

Zulin Zhang ◽

...

Keyword(s):

Polychlorinated Biphenyl ◽

Semen Quality ◽

Sperm Quality ◽

Temporal Trends ◽

Reproductive Function ◽

Environmental Chemicals ◽

Dna Integrity ◽

Diethylhexyl Phthalate ◽

Number Of Males

Abstract Adverse temporal trends in human semen quality and cryptorchidism in infants have been associated with exposure to environmental chemicals (ECs) during development. Here we report that a population of breeding dogs exhibit a 26 year (1988–2014) decline in sperm quality and a concurrent increased incidence of cryptorchidism in male offspring (1995–2014). A decline in the number of males born relative to the number of females was also observed. ECs, including diethylhexyl phthalate (DEHP) and polychlorinated biphenyl 153 (PCB153), were detected in adult dog testes and commercial dog foods at concentrations reported to perturb reproductive function in other species. Testicular concentrations of DEHP and PCB153 perturbed sperm viability, motility and DNA integrity in vitro but did not affect LH stimulated testosterone secretion from adult testis explants. The direct effects of chemicals on sperm may therefore contribute to the decline in canine semen quality that parallels that reported in the human.

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Improving the Rabbit Semen Cryopreservation Protocol: Comparison Between Two Extenders and Inseminating Doses

Annals of Animal Science ◽

10.2478/aoas-2020-0023 ◽

2020 ◽

Vol 20 (3) ◽

pp. 887-898

Author(s):

Michele Di Iorio ◽

Giusy Rusco ◽

Maria Antonietta Colonna ◽

Michele Schiavitto ◽

Maria Silvia D’Andrea ◽

...

Keyword(s):

Liquid Nitrogen ◽

Sperm Motility ◽

Artificial Insemination ◽

Sperm Quality ◽

Dna Integrity ◽

Semen Cryopreservation ◽

Fertilizing Ability ◽

Cryopreservation Protocol

AbstractThis study has been designed to optimize the semen freezing protocol in rabbits, in this regard we compared a Tris-citrate-glucose (TCG) extender with a commercial one (Cortalap®), that to the best of our knowledge has never been used up to now on the in vitro freezability and fertilizing ability of cryopreserved rabbit semen. Two different inseminating semen doses were considered. Five pooled semen samples were divided into two subsamples and each of them were diluted to a ratio 1:1 (v:v) with a freezing extender composed of TCG or Cortalap® containing 16% of dimethylsulfoxide and 0.1 mol/L of sucrose. The extended semen was filled into 0.25 mL plastic straws and frozen above a liquid nitrogen surface. After thawing (50°C/10 seconds) we determined sperm motility, viability, membrane functionality, acrosome and DNA integrity. Our results showed that the Cortalap® extender significantly improved the in vitro post-thaw sperm quality, in comparison to TCG. When we compared the extenders in vivo, no significant differences in the reproductive performances were observed independently by inseminating doses used. In this study we demonstrated that Cortalap® extender can be used as an alternative to TCG. Thus, the Cortalap® being a ready to use extender, implies a reduction of time, mistakes and microbial contaminations during its preparation. This discovery results as significant because it provides beyond an important contribution to the creation of the first Italian semen cryobank of rabbit breeds and also for livestock rabbit farms based on artificial insemination (AI) program.

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Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650190 ◽

1981 ◽

Vol 45 (03) ◽

pp. 290-293 ◽

Cited By ~ 9

Author(s):

Peter H Levine ◽

Danielle G Sladdin ◽

Norman I Krinsky

Keyword(s):

Superoxide Dismutase ◽

Cytochrome C ◽

Xanthine Oxidase ◽

Direct Effect ◽

Platelet Function ◽

Experimental Conditions ◽

Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

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