191 SexedULTRA™, A NEW METHOD OF PROCESSING SEX SORTED BOVINE SPERM IMPROVES POST-THAW SPERM QUALITY AND IN VITRO FERTILITY

Since the first publications 30 years ago showing that flow cytometry was a reliable method to separate X and Y chromosome bearing sperm, the process has been subject to continual refinement. Numerous experiments have been performed in the last few years with the objective of developing an improved sex-sorted product, branded SexedULTRA™ (Sexing Technologies, Navasota, TX, USA) that retains sperm integrity to improve post-thaw sperm quality, in vitro embryo production, and field fertility compared with the previous XY method. Laboratory evaluations were performed on semen from 12 bulls at the Sexing Technologies laboratory in Navasota (TX, USA). Each ejaculate was divided in 2 aliquots and then processed in 1 of 2 methods (XY method or SexedULTRA™). Post-thaw sperm motilities were classified into percent total and progressively motile after thawing (0 h) and after a 3-h incubation at 37°C using a computer-assisted sperm motility analyzer (Hamilton Thorne IVOS II system, Hamilton Thorne Biosciences, Beverly, MA, USA). Percent intact acrosomes was also estimated after a 3-h incubation. Results were analysed by a mixed model ANOVA with the fixed effect of treatment and random effect of bull. Percent total motile SexedULTRA™ sperm was greater (P < 0.001) than sperm processed following the XY method at 0 (78.8 v. 67.2%) and 3 h (51.0% v. 39.0%) post-thaw. Likewise, there was a higher percent of progressively motile sperm both at 0 (50.7 v. 44.9%) and 3 h (31.5 v. 4.4%) post-thaw in the SexedULTRA™ sperm. Percent intact acrosomes was also greater in SexedULTRA™ sperm compared with the sperm processed following previous method (78.0 v. 64.0%). In vitro fertilizations were performed as a measure of sperm competence using 8 ejaculates from 2 bulls in Sexing Technologies IVF laboratory in Laceyville (PA, USA). Five to 10 oocytes and 5,000 motile sperm/oocyte were placed per IVF drop for the analysis. A total of 3 straws and a minimum of 800 oocytes per treatment group (ejaculate × treatment) were included in the comparison for development to 8-cell stage (cleavage rate) and to Day 7 blastocyst stage, measured as total (grades 1 to 4) and freezable (grades 1 and 2) embryos. Results were analysed using a mixed model ANOVA with treatment as a fixed effect and bull, ejaculate within bull, and IVF cycle as random effects. Results from IVF trials are shown in Table 1. Total and freezable embryo numbers were significantly higher (P < 0.05) when using SexedULTRA™ compared with XY sperm. Maintaining a suitable environment for sperm to progress through the various steps of the sex-sorting process results in better semen quality post-thaw as well as improved in vitro fertility. The SexedULTRA™ method confers a significant benefit in maintaining sperm integrity that, if translated into field fertility, could reduce the conception rate gap between conventional and sex-sorted bovine sperm. Table 1. Results from IVF and embryo culture using frozen-thawed, sex-sorted semen processed using the XY or the SexedULTRA™ method

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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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Mitochondria Content and Activity Are Crucial Parameters for Bull Sperm Quality Evaluation

Antioxidants ◽

10.3390/antiox10081204 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1204

Author(s):

Zofia E. Madeja ◽

Marta Podralska ◽

Agnieszka Nadel ◽

Marcin Pszczola ◽

Piotr Pawlak ◽

...

Keyword(s):

Quality Evaluation ◽

Semen Quality ◽

Sperm Quality ◽

Transcript Level ◽

Glutathione Synthetase ◽

Motile Sperm ◽

Bovine Sperm ◽

Cryopreserved Sperm ◽

Group 2 ◽

Group 1

Standard sperm evaluation parameters do not enable predicting their ability to survive cryopreservation. Mitochondria are highly prone to suffer injuries during freezing, and any abnormalities in their morphology or function are reflected by a decline of sperm quality. Our work focused on describing a link between the number and the activity of mitochondria, with an aim to validate its applicability as a biomarker of bovine sperm quality. Cryopreserved sperm collected from bulls with high (group 1) and low (group 2) semen quality was separated by swim up. The spermatozoa of group 1 overall retained more mitochondria (MitoTrackerGreen) and mtDNA copies, irrespective of the fraction. Regardless of the initial ejaculate quality, the motile sperm contained significantly more mitochondria and mtDNA copies. The same trend was observed for mitochondrial membrane potential (ΔΨm, JC-1), where motile sperm displayed high ΔΨm. These results stay in agreement with transcript-level evaluation (real-time polymerase chain reaction, PCR) of antioxidant enzymes (PRDX1, SOD1, GSS), which protect cells from the reactive oxygen species. An overall higher level of glutathione synthetase (GSS) mRNA was noted in group 1 bulls, suggesting higher ability to counteract free radicals. No differences were noted between basal oxygen consumption rate (OCR) (Seahorse XF Agilent) and ATP-linked respiration for group 1 and 2 bulls. In conclusion, mitochondrial content and activity may be used as reliable markers for bovine sperm quality evaluation.

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190 SexedULTRA™, A NEW METHOD OF PROCESSING SEX-SORTED BOVINE SPERM IMPROVES CONCEPTION RATES

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab190 ◽

2017 ◽

Vol 29 (1) ◽

pp. 203 ◽

Cited By ~ 12

Author(s):

R. W. Lenz ◽

C. Gonzalez-Marin ◽

T. B. Gilligan ◽

J. M. DeJarnette ◽

M. D. Utt ◽

...

Keyword(s):

Field Trial ◽

Fixed Effects ◽

Mixed Model ◽

Random Effect ◽

The United States ◽

Conception Rate ◽

Bovine Sperm ◽

Mixed Model Anova ◽

Weighted Means ◽

Conception Rates

The gap in fertility between conventional and sex-sorted bovine sperm, known to be on the order of 10 percentage points, has never been bridged, even by increasing the number of sex-sorted sperm per inseminate. A concerted effort in the last few years has resulted in substantial changes in all stages of the sex-sorting process to develop an improved sex-sorted product called SexedULTRA™ (Sexing Technologies, Navasota, TX, USA). In vitro tests show that SexedULTRA™ maintained sperm integrity better than the previous XY method, and field trials were conducted to determine if this translated into improved bovine field fertility. The initial field trial was performed using ejaculates from 8 Holstein bulls located at Select Sires in Plain City (OH, USA). Each ejaculate was split in 2 aliquots that were then processed following the 2 methods (XY method or SexedULTRA™). A total of 6,930 Holstein heifers were inseminated across 41 commercial herds in the United States. Conception rate data were analysed using mixed model ANOVA (JMP 10.0.0; SAS Institute Inc., Cary, NC) with fixed effects of treatment, bull, bull by treatment interaction, and the random effect of herd. Sex-sorted bull sperm following the SexedULTRA™ method resulted in a greater (P < 0.001) conception rate compared with the XY method sperm (45.7 ± 1.7 v. 41.2 ± 1.6%). This is the first report of an improvement in conception rates using sex-sorted bovine semen in a decade since it became commercially available. A second field trial to investigate dose rate effects and compare against conventional semen was performed using 5 dairy bulls located at German Genetics International GmbH in Cloppenburg (Germany). Each ejaculate was split 4 ways, sex-sorted, and frozen in 0.25-mL straws as follows: XY 2.1 million/straw, SexedULTRA™ 2.1 million/straw, SexedULTRA™ 3.0 million/straw, and SexedULTRA™ 4.0 million/straw. A total of 7,855 heifers were inseminated with these sex-sorted straws, whereas 62,398 heifers were inseminated with conventional straws (15 million/straw) produced using contemporary ejaculates from the same bulls. The 56-day nonreturn rate was calculated by sire and treatment combination, and assigned a weight based on the total number of AI for each combination thereof. Table 1 contains the nonreturn rate weighted means and the relative fertility compared to conventional sperm. Data were analysed using a mixed model ANOVA (JMP 10.0.0; SAS Institute Inc.) with treatment and bull as fixed effects. SexedULTRA™ 4.0 sperm presented the greatest (P < 0.001) 56-day nonreturn rate followed by conventional 15.0 (66.73 v. 65.66%); XY 2.1 presented the lowest (P < 0.001) nonreturn rate. This is the first time a dose response effect with sex-sorted bovine sperm and parity in conception rates with conventional semen has been demonstrated. Table 1. Field trial results for number of inseminations, 56-day nonreturn rate (NRR) weighted means, and relative fertility1 compared with conventional sperm

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Factors affecting storage of Slovak native rabbit semen in the gene bank

Zygote ◽

10.1017/s0967199417000454 ◽

2017 ◽

Vol 25 (5) ◽

pp. 592-600

Author(s):

Barbora Kulíková ◽

Marta Oravcová ◽

Andrej Baláži ◽

Peter Supuka ◽

Peter Chrenek

Keyword(s):

Plasma Membrane ◽

Liquid Nitrogen ◽

Semen Quality ◽

Sperm Quality ◽

Objective Assessment ◽

Membrane Integrity ◽

Computer Assisted ◽

Quality Traits ◽

Plasma Membrane Integrity

SummaryIn this study, fresh and frozen–thawed semen of Nitra and Zobor rabbit breeds were evaluated for potential inter-breed or inter-male differences in sperm quality traits. Individual male semen from four rabbits of each breed were diluted (v:v; 1:1) in a freezing medium composed of a commercial diluent, 16% of dimethyl sulphoxide (DMSO), 4% of Ficoll 70 and 2% of sucrose and frozen in liquid nitrogen vapours before being plunged into liquid nitrogen. Different motility traits, viability and plasma membrane integrity of fresh and frozen–thawed semen were evaluated in vitro using computer-assisted sperm analysis and flow cytometry. To evaluate the sperm fertilization ability, artificial insemination of fresh and frozen–thawed sperm was performed. Our results showed the effect of breed (P ≤ 0.05) on frozen–thawed sperm viability and plasma membrane integrity. Moreover, individual variability in semen quality among the rabbits was revealed (0.31 to 0.71 among quality traits). Our results thereby confirmed that the cryopreservation procedure could not ensure comparable sperm post-thaw survival for different breeds or males. Nevertheless, correlations between numbers of fresh total motile and progressively moving sperm and several quality parameters measured post thawing were revealed. Therefore, we suggest that the objective assessment of fresh rabbit sperm motility may be an effective indicator of frozen–thawed semen quality. Consequently, regular semen assessment is required in order to preserve good-quality insemination doses from native breeds.

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Post-wash total motile sperm count a useful predictor in the decision to perform IVF/ICSI in patients with non-male factor infertility

Journal of Shifa Tameer-e-Millat University ◽

10.32593/jstmu/vol3.iss2.108 ◽

2020 ◽

Vol 3 (2) ◽

pp. 99-106

Author(s):

Sara Mahmood Qureshi ◽

Salma Kafeel ◽

Riffat Bibi ◽

Jawad Mohmand

Keyword(s):

Sperm Quality ◽

Sperm Count ◽

Adverse Outcomes ◽

Male Factor Infertility ◽

Motile Sperm ◽

Operating Characteristics ◽

Male Factor ◽

Vitro Fertilization ◽

Total Motile Sperm Count

Introduction: The unrestricted use of intracytoplasmic sperm injection (ICSI) for non-male factor infertility is associated with adverse outcomes. Post-wash total motile sperm count (PW-TMSC) offers prognostic value to assess sperm quality and aid in the decision to perform in vitro fertilization (IVF) or ICSI. Objectives: The aim of this study was to identify the effect of PW-TMSC on fertilization rates in patients undergoing IVF cycles exclusively with non-male factor infertility. It also aimed to identify whether unnecessary ICSI could be avoided in such cases, thus maximizing optimal outcomes. Materials & Methods: We retrospectively analyzed age, semen volume, prewash TMSC, and PW-TMSC in 68 conventional IVF cycles of infertile couples with non-male factor infertility. Clinical characteristics including female age, number of follicles, level of estradiol on trigger day, mature cumulus-oocyte complexes (COCs) collected, were also included. Results: Incidence of <30% fertilization was significantly higher in the 4-<10 Million group compared with the ≥20 Million post-wash TMSC group (P<0.001). Furthermore, Receiver operating characteristics (ROC) analysis revealed post-wash TMSC as a significant predictor (P<0.05) of total failed fertilization (TFF) and of ≥30% fertilization (P<0.05) with area under curve (AUC) of 0. 79 and 0.77, respectively, with a deemed cutoff of 10.89 Million. Conclusion: Post-wash TMSC is a good predictor of fertilization; it can help in avoiding potentially low or even total fertilization failure (TFF). A cut-off point of 10.89 Million or less should warrant the use of ICSI.

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Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer

Zygote ◽

10.1017/s096719941600037x ◽

2016 ◽

Vol 25 (1) ◽

pp. 85-97 ◽

Cited By ~ 7

Author(s):

María Elena Arias ◽

Esther Sánchez-Villalba ◽

Andrea Delgado ◽

Ricardo Felmer

Keyword(s):

Gene Transfer ◽

Sperm Quality ◽

Computer Assisted ◽

Exogenous Dna ◽

Sperm Analysis ◽

Functional Parameters ◽

Transgenic Embryos ◽

Fertilization Capacity ◽

Motility Parameters

SummarySperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) orin vitrofertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

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105 Increasing fertility of interspecific hybrid males using biotechnological approaches

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab105 ◽

2021 ◽

Vol 33 (2) ◽

pp. 159

Author(s):

A. Vetokh ◽

A. Tadzhieva ◽

B. Iolchiev ◽

N. Volkova ◽

V. Bagirov

Keyword(s):

Dna Fragmentation ◽

Interspecific Hybrid ◽

Semen Quality ◽

Sperm Quality ◽

Semen Analysis ◽

Differential Staining ◽

Computer Assisted ◽

Sperm Dna Fragmentation ◽

Russian Science

The results of AI depend on many factors, with the quality of semen being one of the most important. Not all male hybrids can meet the requirements for semen quality, because they often have reduced fertility following cryopreservation. Thus, it is necessary to improve semen processing before use in AI. The aim of the study was to evaluate the effectiveness of using the “swim-up” flotation method to improve sperm quality of hybrid males of the Ovis genus. Semen from interspecific hybrid rams (1/4 Argali×3/4 Romanov, n=15; 1/8 Argali×7/8 Romanov, n=15) was freshly obtained, frozen–thawed, and processed by the swim-up method. Evaluation of sperm motility was determined using computer-assisted semen analysis. Statistical analysis was performed using SPSS vs.15.0 (ANOVA and t-test; SPSS Inc.). Semen was collected during the breeding season (October–December) via artificial vagina. Assessment of acrosome integrity was determined using differential staining with a Diachem diff-quick kit (NPF ABRIS+). The degree of sperm DNA fragmentation was determined using the acridine-orange test. The sperm freezing/thawing cycle was accompanied by sperm damage and an increase in the proportion of immobile sperm from 10 to 58%, with non-progressive movement increasing from 9 to 19.3%. The number of spermatozoa with abnormal morphology doubled, and the DNA fragmentation index increased from 16 to 26%. Use of the swim-up procedure allowed us to sort progressively motile spermatozoa. The content of progressively motile spermatozoa in the samples obtained from the supernatant was 86%, which was 2.3 times higher than in frozen–thawed sperm (P≤0,01). The obtained results show the effective use of the swim-up procedure to determine the quality of semen in hybrid rams. These studies were carried out with financial support from the Russian Science Foundation, grant No. 18-16-00079 and the Ministry of Science and Higher Education of the Russian Federation.

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23 Sperm quality of Pure Spanish stallions is affected by inbreeding coefficient and age

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab23 ◽

2020 ◽

Vol 32 (2) ◽

pp. 137

Author(s):

Y. Pirosanto ◽

M. Valera ◽

A. Molina ◽

J. Dorado ◽

S. Demyda-Peyrás

Keyword(s):

Sperm Motility ◽

Inbreeding Depression ◽

Negative Correlation ◽

Semen Quality ◽

Sperm Quality ◽

Sperm Concentration ◽

Inbreeding Coefficient ◽

Semen Parameters ◽

Computer Assisted

Inbreeding depression, a genetic condition produced by the mating of close-related individuals, has been associated with a reduction of fertility in several species. However, a loss in sperm quality was also associated with age. In horses, the few existing reports have described a tendency of both parameters to produce a negative effect on sperm quality. However, those reports were performed using a subjective evaluation of sperm motility. In the present study, a total of 692 ejaculates from 86 Pure Spanish stallions (PRE), aged between 3 and 22 years, were evaluated using a computer-assisted methodology to determine the effect of inbreeding in four semen parameters: free-gel volume (V), sperm concentration (C, by haemocytometer), and total (TM) and progressive (PM) sperm motility (by Spermvision sperm class analyser; Minitube). The inbreeding coefficient (F) was estimated using 300 000 PRE pedigree records approximately (minimum pedigree depth, eight equivalent complete generations; range, between 1 and 30.1%). Stallion, age, ejaculate, and season of semen collection were the variables included in the statistical model (general linear model), with ejaculate and season being the variables with a major effect (by variance components analysis). Our results showed that sperm concentration (r=−0.18; P<0.0001) and volume (to a lesser extent) were reduced with advancing age, both showing a major decline after 15 years of age. To the contrary, sperm motility was not affected by age of the stallion. We also found a negative correlation between the inbreeding coefficient and ejaculate volume (r=−0.14; P<0.001), with a marked decrease seen when F was between 7 and 20%. Also, a negative correlation was observed in PM (r=−0.08; P<0.05), although to a lower extent. Conversely, C and TM were not affected by inbreeding depression (P>0.05). In conclusion, our results demonstrated that high levels of inbreeding can compromise severely the sperm quality of the PRE stallion, which, subsequently, may have a negative influence on fertility. Ongoing studies using genomic data will help to detect genetic variants associated with stallion semen quality and how it is influenced by inbreeding in specific genomic regions.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21228659 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8659

Author(s):

Maria Pachetti ◽

Luisa Zupin ◽

Irene Venturin ◽

Elisa Mitri ◽

Rita Boscolo ◽

...

Keyword(s):

Lipid Composition ◽

Semen Quality ◽

Sperm Quality ◽

Sample Selection ◽

Poor Quality ◽

Interesting Possibility ◽

The Many ◽

Α Helix ◽

Analytical Approaches

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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