FTIR Spectroscopy to Reveal Lipid and Protein Changes Induced on Sperm by Capacitation: Bases for an Improvement of Sample Selection in ART

Although being a crucial step for Assisted Reproduction Technologies (ART) success, to date sperm selection is based only on morphology, motility and concentration characteristics. Considering the many possible alterations, there is a great need for analytical approaches allowing more effective sperm selections. The use of Fourier Transform Infrared (FTIR) may represent an interesting possibility, being able to reveal many macromolecular changes in a single measurement in a nondestructive way. As a proof of concept, in this observational study, we used a FTIR approach to reveal features related to sperm quality and chemical changes promoted by in vitro capacitation. We found indication that α-helix content is increased in capacitated sperm, while high percentages of the β-structures seem to correlate to poor-quality spermatozoa. The most interesting observation was related to the lipid composition, when measured as CH2/CH3 vibrations (ratio 2853/2870), which resulted in being strongly influenced by capacitation and well correlated with sperm motility. Interestingly, this ratio is higher than 1 in infertile samples, suggesting that motility is related to sperm membranes stiffness and lipid composition. Although further analyses are requested, our results support the concept that FTIR can be proposed as a new smart diagnostic tool for semen quality assessment in ART.

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Fructose content and fructolysis in semen. Practical application in the evaluation of semen quality

The Journal of Agricultural Science ◽

10.1017/s0021859600006109 ◽

1948 ◽

Vol 38 (3) ◽

pp. 323-331 ◽

Cited By ~ 109

Author(s):

T. Mann

Keyword(s):

Semen Quality ◽

Poor Quality ◽

Small Samples ◽

Bull Semen ◽

Accessory Glands ◽

Reduced Rate ◽

High Level ◽

Fresh Semen

SUMMARY1. A method is described whereby fructose content and fructolysis can be assayed accurately in small samples of semen. The advantages of this method lie in its simplicity, accuracy and practical convenience as a tool for the assessment of semen quality, applicable also under field conditions.2. The content of fructose in fresh semen depends upon the secretory function of accessory glands which is influenced directly by the activity of the male sex hormone. A low level of seminal fructose may coincide with other symptoms of hormonal malfunction and poor quality of spermatozoa. A high level of seminal fructose indicates satisfactory functional ability of the accessory glands, but it does not necessarily coincide with high quality of spermatozoa as expressed in terms of density and motility.3. The normal level of fructose in fresh semen undergoes frequent fluctuations which can be observed if semen collections are made from the same individual at different times. Considerable variations in the sperm/fructose ratio may also occur in the semen of the same individual as illustrated by the results of an ‘exhaustion test’.4. Fructose disappears from semen incubated in vitro. The rate of fructose disappearance forms a convenient measure of sperm fructolysis. The normal rate of fructolysis in bull semen is 1·4–2 mg. fructose per 109 sperm cells in 1 hr. at 37° C. At this high level it can be maintained until almost the whole reserve of fructose has been exhausted. Azoospermic and necrospennic semen, as well as that from vasectomized animals, are unable to utilize fructose. A reduced rate of fructolysis is found in low quality semen of subfertile and infertile animals.5. The conditions of sperm survival in semen incubated in narrow tubes as used for the fructolysis test as well as for storage of semen in the practice of artificial insemination, are almost purely anaerobic. Under such conditions the survival of spermatozoa must largely depend upon fructolysis and not upon respiration.

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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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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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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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180 ASSESSMENT OF BULL SEMEN QUALITY LOADED IN NEW SensiTemp STRAWS USING SEMEN AND IN VITRO PRODUCTION TECHNOLOGIES

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab180 ◽

2016 ◽

Vol 28 (2) ◽

pp. 221

Author(s):

D. Le Bourhis ◽

S. Camugli ◽

P. Salvetti ◽

L. Schibler ◽

E. Schmitt

Keyword(s):

Sperm Motility ◽

Semen Quality ◽

Sperm Quality ◽

Semen Analysis ◽

Membrane Integrity ◽

Computer Assisted ◽

Cleavage Rate ◽

Fluid Medium ◽

And Control

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liège, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P < 0.05). In experiment 1, neither sperm motility from bull 1 (61.2 and 60.5%) and bull 2 (66.2 and 66.5%) nor membrane integrity from bull 1 (58.6 and 52.2%) and bull 2 (61.0 and 61.9%) were different between SensiTemp and control, respectively. Results from experiment 2 showed no difference (P > 0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P > 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.

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Season-induced variation in lipid composition is associated with semen quality in Holstein bulls

Reproduction ◽

10.1530/rep-12-0498 ◽

2013 ◽

Vol 145 (5) ◽

pp. 479-489 ◽

Cited By ~ 24

Author(s):

N Argov-Argaman ◽

K Mahgrefthe ◽

Y Zeron ◽

Z Roth

Keyword(s):

Fatty Acid ◽

Lipid Composition ◽

Semen Quality ◽

Sperm Quality ◽

Structural Parameters ◽

Lipid Extraction ◽

Sperm Head ◽

High Saturated Fatty Acid ◽

Seminal Fluids ◽

Induced Variation

Season-induced variation in fatty acid and cholesterol composition in bovine semen has been associated with semen quality. Given the specific roles of the various semen compartments (seminal fluids, sperm head, and sperm tail) in fertilization, we hypothesized that environmental-stress-induced alterations in the lipid composition of a specific compartment might impair semen quality and sperm function. Semen samples were collected from five mature Holstein–Friesian bulls during the summer (August to September) and winter (December to January). Semen was evaluated by computerized sperm-quality analyzer, calibrated for bulls' semen, and centrifuged to separate the spermatozoa from the seminal fluids. The spermatozoal fraction was sonicated to separate the sperm head and tail compartments. Cold lipid extraction was performed with chloroform:methanol (2:1, vol/vol). Lipids were identified and quantified by gas chromatography. Seasonal variation was found in both physiological and structural parameters. The proportion of spermatozoa defined as morphologically normal was higher in the winter, with higher motility, progressive motility, and velocity relative to summer samples. Lipid composition within fractions varied between seasons with prominent impairment in the tail compartment, characterized by high saturated fatty acid, low polyunsaturated fatty acid, and low cholesterol concentrations during the summer. Given the association between alterations in lipid composition and reduced sperm motility and velocity during the summer, it is suggested that lipid composition might serve to predict sperm quality.

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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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Respiratory Mitochondrial Efficiency and DNA Oxidation in Human Sperm after In Vitro Myo-Inositol Treatment

Journal of Clinical Medicine ◽

10.3390/jcm9061638 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1638 ◽

Cited By ~ 1

Author(s):

Laura Governini ◽

Rosetta Ponchia ◽

Paolo Giovanni Artini ◽

Elena Casarosa ◽

Ilaria Marzi ◽

...

Keyword(s):

Dna Damage ◽

Oxidative Phosphorylation ◽

Oxidative Damage ◽

Sperm Motility ◽

Semen Quality ◽

Sperm Quality ◽

Antioxidant Properties ◽

Atp Production ◽

In Vitro Treatment

Semen samples are known to contain abnormal amounts of reactive oxygen species (ROS) and oxygen free radicals; therefore, the identification of antioxidant molecules able to counteract the oxidative damage caused by ROS is foresight. Indeed, improving semen quality in terms of motility and reduction in DNA damage, can significantly improve the fertilization potential of sperm in vitro. To this regard, myo-inositol, based on its antioxidant properties, has been reported to be effective in improving sperm quality and motility in oligoasthenozoospermic patients undergoing assisted reproduction techniques when used as a dietary supplementation. Moreover, in vitro treatment demonstrated a direct relationship between myo-inositol, mitochondrial membrane potential and sperm motility. This experimental study aimed to evaluate the effects of myo-inositol (Andrositol-lab) in vitro treatment on sperm motility, capacitation, mitochondrial oxidative phosphorylation and DNA damage. Our results demonstrate that myo-inositol induces a significant increase in sperm motility and in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production, both in basal and in in vitro capacitated samples. Moreover, we provide evidence for a significant protective role of myo-inositol against oxidative damage to DNA, thus supporting the in vitro use of myo-inositol in assisted reproductive techniques. Even if further studies are needed to clarify the mechanisms underlying the antioxidant properties of myo-inositol, the present findings significantly extend our knowledge on human male fertility and pave the way to the definition of evidence-based guidelines, aiming to improve the in vitro procedure currently used in ART laboratory for sperm selection.

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Study on the in vitro fertilizing potential of mithun (Bos frontalis) semen

Indian Journal of Animal Research ◽

10.18805/ijar.11323 ◽

2016 ◽

Author(s):

P. Perumal ◽

S. K. Srivastava ◽

K. K. Baruah ◽

S. K. Ghosh

Keyword(s):

Semen Quality ◽

Egg Yolk ◽

Forward Direction ◽

Poor Quality ◽

Quality Parameters ◽

The Poor ◽

Acrosomal Membrane ◽

Sperm Abnormality ◽

Biochemical Profiles

Semen quality parameters (SQPs) such as motility, viability, total sperm abnormality, integrity of acrosome, plasma membrane and nucleus and vanguard distance travelled by sperm and biochemical profiles such as aspartate amino transaminase (AST), alanine amino transaminase (ALT) and lactate dehydrogenase (LDH) were studied in mithun at post thawed stage. Fifty ejaculates were collected through transrectal massage method from matured bulls and the ejaculates were divided into good and poor quality. The ejaculates were extended with Tris egg yolk citrate glycerol extender and stored in liquid nitrogen. PT seminal, biochemical profiles and zona binding per cent (BP) and binding index (BI) were studied with standard protocols. The results revealed good quality ejaculates has significantly (p<0.05) higher SQPs, BP & BI and has significantly (p<0.05) lower total sperm abnormality, AST, ALT and LDH than in the poor quality ejaculates. PT motility was positively (p<0.05) correlated with SQPs, vanguard distance, BI & BP and negatively correlated (p<0.05) with total sperm abnormality and biochemical profiles. In conclusion, the good quality sperm has higher functional sperm structures and higher intactness of sperm plasma and acrosomal membrane to motile faster in forward direction to reach the site of fertilization and successful fertilization.

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Hyperviscous Semen Causes Poor Sperm Quality and Male Infertility through Induction of Oxidative Stress

Current Urology ◽

10.1159/000499302 ◽

2019 ◽

Vol 13 (1) ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Asghar Beigi Harchegani ◽

Hamid Rahmani ◽

Eisa Tahmasbpour ◽

Alireza Shahriary

Keyword(s):

Oxidative Stress ◽

Male Infertility ◽

Molecular Mechanisms ◽

Semen Quality ◽

Sperm Quality ◽

Fertilization Rate ◽

Cellular Mechanisms ◽

Vitro Fertilization ◽

Randomized Controlled Studies

Background/Aims: Semen hyperviscosity (SHV) is one of the significant factors involved in poor semen quality and male infertility. It also leads major problems during assisted reproduction techniques and in vitro fertilization process. Although influence of SHV on sperm quality, fertilization rate and male infertility have been widely considered, molecular and cellular mechanisms for these abnormalities are not well understood. In this review, we aimed to discuss the proposed cellular and molecular mechanisms of SHV on male reproductive system, the importance of oxidative stress (OS) and the mechanisms by which SHV induces OS and impairment of other antioxidants. Methods: A PubMed/Medline and EM-BASE search was performed using keywords: “hyperviscosity semen”, “oxidative stress”, and “male infertility”. Conclusion: OS induced by reactive oxygen species can be considered as a major mechanism in patients with hyperviscosity semen that is associated with DNA fragmentation, lipid peroxida-tion and sperm membrane disintegrity, apoptosis, depletion of antioxidants, and subsequently poor sperm quality and male infertility. Therefore, antioxidant therapy may improve main pathological effects of hyperviscosity semen, especially oxidative damages and inflammation, on sperm quality and function. Further, randomized controlled studies are necessary to confirm these results and make a comparison between effects of various antioxidants such as N-acethyl-cysteine and Curcumin on fertility problem in patients with hyperviscous semen.

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