68 Comparison of two Percoll gradients for selection of frozen semen for invitro production of ovine embryos

2021 ◽  
Vol 33 (2) ◽  
pp. 141
Author(s):  
A. Velázquez-Roque ◽  
F. Villaseñor-González ◽  
G. Márquez-Márquez ◽  
M. Kjelland ◽  
H. Álvarez-Gallardo ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Physiological Saline ◽  
Bottom Layer ◽  
Penicillin G ◽  
Sperm Selection ◽  
Percoll Gradient ◽  
Cleavage Rate ◽  
Frozen Semen ◽  
Percoll Gradients ◽  
Α Level

Sperm selection methods are routinely applied to prepare semen for IVF in animal species. These procedures are used to improve sperm quality characteristics as well as to remove other background material and debris. Percoll gradient is widely used in animal IVF laboratories. Different Percoll gradient concentrations and volumes can be used to improve the sperm sample motility percentage. The objective of this research was to evaluate the effect of 2 different Percoll concentrations for ovine IVF sperm selection and effects, if any, on invitro embryo production (IVP). Specifically, Percoll gradients consisted of Group 1 (G1) 40–80% and Group 2 (G2) 45–90%, 400µL each. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries (n=157) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IU mL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=800, divided equally into 4 replicates) were divided into 2 groups, G1 and G2. The IVF process was conducted with semen selected through a mini-Percoll technique with gradients at a concentration of 45% (top layer) and 90% (bottom layer) or 40% (top layer) and 80% (bottom layer) for G1 and G2, respectively, using frozen–thawed semen from the same ram, at a concentration of 2×106 sperm mL−1, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The percentages of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated, based on the initial number of oocytes entering into IVM. The statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of G1 versus G2 (α level=0.05). Cleavage rate was 47.8%±2.5 and 55.9%±2.5, respectively, in G1 and G2. The percentages of embryos with more than 6 cells were 38.1%±2.2 and 43.5%±2.2, respectively, in G1 and G2. The percentage of blastocysts on Day 7 was 27.4%±1.1 and 37.3%±1.1, respectively, in G1 and G2. There were no significant differences between groups (P>0.05) for percentage of cleavage and embryos with more than 6 cells, although the percentage of cleavage tended to be greater for G2 (P=0.06). Additionally, G2 had a larger percentage of blastocysts on Day 7 compared with G1 (P<0.05). In conclusion, under the conditions of this research, the use of a Percoll gradient at a concentration of 40–80% increased the percentage of blastocysts for ovine IVP.

51 Comparison of SexedULTRA 4M and conventional semen for invitro production of bovine embryos using two bulls

2021 ◽  
Vol 33 (2) ◽  
pp. 132
Author(s):  
H. Álvarez-Gallardo ◽  
M. Kjelland ◽  
M. Pérez-Martínez ◽  
F. Villaseñor-González ◽  
S. Romo
Keyword(s):  
Physiological Saline ◽  
Penicillin G ◽  
Bovine Embryos ◽  
Streptomycin Sulphate ◽  
Cleavage Rate ◽  
Cattle Industry ◽  
Sexed Semen ◽  
Physiological Saline Solution ◽  
Sorting Process ◽  
Α Level

SexedULTRA 4M™ (Select Sires) is made using an improved method of sex-sorting sperm in a less damaging environment for retaining sperm integrity through the sorting process. The combination of invitro embryo production (IVP) and sexed semen technologies has been successful for intensity selection in the cattle industry. The objective of this research was to compare conventional (CONV) and SexedULTRA 4M (ULTRA-4M) semen for bovine IVP using 2 bulls. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries from commercial cattle (n=213) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IUmL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For the IVM, the cumulus–oocyte complexes (COCs) were selected (grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=1200, divided equally into 3 replicates) were divided into 2 groups, the CONV group and the ULTRA-4M group. The IVF process was conducted with both CONV and ULTRA-4M semen from the 2 bulls, separately, at an adjusted concentration of 2×106 and 0.5×106 sperm mL−1, respectively, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2 and 90% N2 at 100% humidity. The cleavage rate, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated. Statistical analysis was carried out with the GLM procedure of SAS (version 9.3; SAS Institute Inc.) to evaluate the results of CONV versus ULTRA-4M for each bull (α level=0.05). Cleavage rates were 57.1%±1.5 and 59.4%±1.5, respectively in CONV and ULTRA-4M groups with Bull 1 and 43.3%±1.5 and 45.2%±1.5 with Bull 2. The percentages of embryos with more than 6 cells were 51.4%±1.0 and 53.9%±1.0, respectively, with CONV and ULTRA-4M with Bull 1 and 30.4%±1.0 and 33.4%±1.0 with Bull 2. The percentage of blastocysts on Day 7 with Bull 1 was 34.4%±1.7 for CONV and 36.2%±1.7 for ULTRA-4M; for Bull 2, the results were 26.5%±1.7 for CONV and 29.5%±1.7 for ULTRA-4M. There were no significant differences between the CONV and ULTRA-4M groups (P>0.05) for all variables analysed for each bull; however, Bull 1 was significantly superior to Bull 2 for all variables analysed. In conclusion, under the conditions of this research, ULTRA-4M and CONV semen produced similar bovine IVP results.

scholarly journals Use of a colloid to optimize centrifugation in the selection of bovine sperm for IVF

2018 ◽  
Vol 39 (4) ◽  
pp. 1607 ◽  
Author(s):  
Cibele Garcia Moreira Gonçalves ◽  
Fábio Gallas Leivas ◽  
Daniele Missio ◽  
Francielli Weber Santos ◽  
Eduardo Brum Schwengber ◽  
...  
Keyword(s):  
Recovery Rate ◽  
Sperm Quality ◽  
Ros Production ◽  
Sperm Selection ◽  
Percoll Gradient ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
The Impact ◽  
Normal Fertilization

This study aimed to evaluate the effect of the force and duration of centrifugation and the impact of cushioned centrifugation on sperm selection by Percoll gradient, on sperm quality and development kinetics of in vitro produced bovine embryos. Two experiments were performed. In Experiment I, a pool of semen was selected by Percoll gradients and the pellet was divided into four groups and distributed in a 2 × 2 factorial, with two forces (2200 × g or 9000 × g) and two durations (1 min or 3 min) of centrifugation. In Experiment II, semen was divided into two groups and selected by Percoll gradient with Cushion Fluid (CF) or without CF (Control) in the second centrifugation. The morphofunctionality, biochemical characteristics and fertilizing capacity of the selected sperms were evaluated. In addition, the development of the resulting bovine embryos was monitored for 48 h post-insemination. Duncan and Chi-square tests (P < 0.05) were used to compare the means. In Experiment I, there was a significant increase in sperm vigor (P < 0.05) after sperm selection in all treatments. The force and duration of centrifugation did not have any effect on sperm motility, vigor, and recovery rate among the different treatments (P > 0.05). In Experiment II, the recovery rate and reactive oxygen species (ROS) production in semen were similar among treatments (P > 0.05) although a higher ROS production was observed in the CF fertilization medium. Total fertilization rate was superior in the CF group (65.4 ± 5.3%) compared to that in Control (39.6 ± 4.9%). However, the normal fertilization and cleavage rate did not differ between the Control (94 ± 6.3% and 58.3 ± 8.3%) and CF (89 ± 7.1% and 75.0 ± 7.3%) groups. The reduction in the force and duration of centrifugation did not decrease the sperm recovery during selection by the Percoll gradient and the use of CF in the second centrifugation did not affect the normal fertilization and development of bovine IVF embryos up to 48 h.

71 Comparison of sexed semen ULTRA-4M with conventional semen for the invitro production of bovine embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 161
Author(s):  
H. Alvarez ◽  
M. Kjelland ◽  
F. Villaseñor ◽  
M. Pérez ◽  
S. Romo
Keyword(s):  
Sperm Concentration ◽  
Physiological Saline ◽  
Field Trials ◽  
Penicillin G ◽  
Cleavage Rate ◽  
Cattle Industry ◽  
The United Kingdom ◽  
Sexed Semen ◽  
Physiological Saline Solution ◽  
Α Level

The first commercial production of sexed semen was at the Cogent company in the United Kingdom. Since then millions of offspring have been born using sexed semen produced by flow cytometry. Sexed semen technology has recently been modernized to what is now known as ULTRA (ST Genetics), completely modifying the technique, medium, and sperm concentration. In field trials using AI, there was no difference between conventional semen (CONV) and ultra-sexed semen at a concentration of 4 million sperm per straw (ULTRA-4M). The combination of invitro embryo production (IVP) and sexed semen technologies has been successful for intensity selection in the cattle industry. The objective of this work was to compare the CONV and ULTRA-4M semen for bovine IVP. The research was carried out in the reproduction laboratory at the Facultad de Estudios Superiores Cuautitlán-Universidad Nacional Autónoma de México (FESC-UNAM). The IVP was performed with a continuous invitro culture (IVC) system. Ovaries (n=213) were collected from a slaughterhouse (Querétaro, México) and transported to the laboratory within 2h (FESC-UNAM) in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IUmL−1) and streptomycin sulfate (100µgmL−1). For IVP, VITROGEN media were used for IVM, IVF, and IVC. For the IVM, the cumulus-oocyte complexes were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2, 95% air, and 100% humidity. Matured oocytes (n=1000) were divided into two groups, the CONV group and the ULTRA-4M group. The IVF process was developed with CONV and ULTRA-4M semen from the same bull (Holstein) at a concentration of 2×106 and 0.5×106 spermmL−1, respectively, for 18h in 38.5°C, 5% CO2, 95% air, and 100% humidity. The presumptive zygotes were denuded by pipetting and left in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The cleavage rate, embryos of more than 6 cells, and blastocysts on Day 7 of culture were evaluated. The statistical analysis was carried out with the GLM procedure of the SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of CONV vs. ULTRA-4M (α level=0.05). Percent cleavage for CONV was 72.2%±2.53 and 75.6%±2.53 for ULTRA-4M. For embryos with more than 6 cells, the results for CONV and ULTRA-4M were 59.8%±5.61 and 62.8%±5.61, respectively. The percentage of blastocysts on Day 7 was 37.8%±5.39 for CONV and 43.6%±5.39 for ULTRA-4M. There were no significant differences between the groups (P&gt;0.05) for all variables analysed. Although the number of blastocysts on Day 7 were numerically higher in the ULTRA-4M, differences were not significant. In conclusion, under the conditions of this research the ULTRA-4M had similar results as CONV for bovine IVP.

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 217
Author(s):  
E. Mellisho ◽  
V. Rivas ◽  
J. Ruiz ◽  
G. Mamani
Keyword(s):  
Extended Period ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Cleavage Rate ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Washing Method

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

69 Invitro production of ovine embryos using either fresh or frozen-thawed semen

2021 ◽  
Vol 33 (2) ◽  
pp. 141
Author(s):  
G. Márquez-Márquez ◽  
A. Velázquez-Roque ◽  
F. Villaseñor-González ◽  
M. Kjelland ◽  
H. Álvarez-Gallardo ◽  
...  
Keyword(s):  
Semen Quality ◽  
Small Ruminants ◽  
Physiological Saline ◽  
Ovis Aries ◽  
Penicillin G ◽  
Domestic Sheep ◽  
Assisted Reproductive Technique ◽  
Physiological Saline Solution ◽  
Α Level ◽  
Fresh Semen

Invitro embryo production (IVP) is an important tool for genetic improvement in small ruminants. Semen quality is one of the most important aspects to consider for the success of this assisted reproductive technique. With ovine IVP, it is a common practice to use fresh semen for IVF, but this could be a problem because the differences between ejaculates from the same animal are well documented and a source of variation in IVP results. The objective of this research was to compare the effect of fresh and frozen–thawed domestic sheep (Ovis aries) semen on IVF for ovine IVP. The research was carried out in the reproduction laboratory at the Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries (n=186) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IU mL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF-Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=1000) were invitro fertilized using either fresh or frozen–thawed semen (Triladyl™; Minitube) from the same sheep, at a concentration of 2×106 sperm mL−1, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The percentages of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated, based on the initial number of oocytes entering into IVM. Statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of fresh versus frozen–thawed (α level=0.05). Rates of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 were similar (P&gt;0.05): fresh 52.3±3.0%, 43.6±2.6%, and 34.3±2.9%, respectively; frozen–thawed: 53.3%±3.0, 41.1±2.6%, and 33.3±2.9%, respectively. In conclusion, under the conditions of this research, the use of fresh and frozen–thawed semen had similar results for ovine IVP.

151 Effect of season on the in vitro fertilizing ability of frozen - thawed bovine spermatozoa

2019 ◽  
Vol 31 (1) ◽  
pp. 200
Author(s):  
M. Sabes-Alsina ◽  
M. Wallgren ◽  
Y. Sjunesson ◽  
N. Lundeheim ◽  
M. López-Béjar ◽  
...  
Keyword(s):  
Embryo Development ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Mitochondrial Potential ◽  
Semen Collection ◽  
Frozen Semen ◽  
Fertilizing Ability

Previous research indicated that the season during which oocytes are harvested affects quality of in vitro-produced embryos (Gupta et al. 2016 Anim. Reprod. Sci. 164, 162). In our own studies, sperm kinematics, membrane integrity, acrosome status, mitochondrial potential and reactive oxygen species were affected by season of semen collection (Sabés-Alsina et al. 2017 Vet. Rec. 180, 251). The aim of this study was to investigate effect of season of semen collection on in vitro fertilizing ability and embryo development of the same sperm samples from the sperm quality study, using lower than normal sperm doses to detect small differences between groups (Ward et al. 2003 Theriogenology 59, 1575). Frozen semen was available from 8 Holstein bulls kept outdoors in northern Spain, collected during winter, spring and summer. Bovine ovaries, Holstein and Swedish Red breeds, were obtained from an abattoir in spring. Oocytes were matured and fertilized in vitro with a low dose of frozen-thawed sperm (sperm:oocyte ratio 2500:1). After fertilization, presumptive embryos were evaluated at 44h for cleavage and on Day 8 for blastocyst development. Number of sperm binding to the zona pellucida and number of nuclei in developing blastocysts were assessed after staining with Hoechst 33342. There were 2 or 3 replicates per bull. For 555 oocytes inseminated, cleavage rates for winter, spring and summer semen collections were 42, 49 and 47%, respectively; blastocyst rates were 7, 12 and 8%; blastocyst cell numbers were 8.7, 10.2 and 7.8; and mean number of sperm bound was 0.70, 0.63 and 0.50. Although there were no differences (P&gt;0.05) due to season of semen collection for any of these end points, individual bulls had considerable variation in cleavage rate. In conclusion, despite previously published differences in sperm quality with season from these bulls, ability of frozen-thawed sperm to fertilize bovine oocytes and initial embryo development in vitro were not affected by season of semen collection, at least for oocytes collected in spring. However, bull-to-bull variation in cleavage rate was high. The authors gratefully acknowledge the financial support of the Ministerio de Economía y Competitividad and FEDER (AGL2016-79802-P). M. Sabes-Alsina was supported by a PIF from the Universitat Autònoma de Barcelona and by the STS of the Epiconcept COST Action; J. M. Morrell was funded by the Swedish Research Council for the environment, agricultural sciences and spatial planning (FORMAS; 221-2010-1241) and the Swedish Farmers’ Association (SLF; 1330039).

scholarly journals Sperm protein markers for Holstein bull fertility at National Artificial Insemination Centers in Indonesia

2020 ◽  
Vol 13 (5) ◽  
pp. 947-955
Author(s):  
Zulfi Nur Amrina Rosyada ◽  
Mokhamad Fakhrul Ulum ◽  
Ligaya I. T. A. Tumbelaka ◽  
Bambang Purwantara
Keyword(s):  
Artificial Insemination ◽  
Sperm Quality ◽  
High Fertility ◽  
Fertility Level ◽  
Sperm Protein ◽  
Sperm Proteins ◽  
Frozen Semen ◽  
Sds Page ◽  
Microscopic Evaluation ◽  
Holstein Bulls

Background and Aim: Holstein cows and heifers are widely bred in Indonesia by artificial insemination (AI) to increase population and milk production. Sperm fertility is modulated by genetic factors, but the analysis of sperm quality is still based on macro- and microscopic characteristics. This study aimed to analyze both sperm quality and proteins of Holstein bulls at different fertility levels. Materials and Methods: The frozen semen samples were collected from the Indonesia National AI Center. They were classified based on the reproductive efficiency data and were grouped into high fertile (HF) and low fertile (LF). Sperm qualities were evaluated by microscopic evaluation. The Holstein sperm proteins were extracted using phenylmethanesulfonyl fluoride as a protease inhibitor and the benzidine detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to analyze the molecular weights (MWs) of the sperm proteins. The data obtained were analyzed by a t-test using the one-factor bull fertility level, and Spearman's correlation analysis was used to identify the correlation between the sperm microscopic evaluation parameters and protein bands. Results: The sperm motility post-freeze thawing was not significantly different between the HF and LF (p>0.05). The HF level had a higher percentage of viability, intact plasma membrane integrity, and intact acrosomes than the LF (p<0.05). Five protein bands were found in the SDS-PAGE of sperm proteins of Holstein bulls with different concentrations. Sperm proteins with MWs of 17.51 kDa, 14.87 kDa, 33.71 kDa, and 41.97 kDa were abundant in the Holstein bulls with an HF level, while 55 kDa proteins were abundant in the LF level of Holstein bulls. The sperm of Holstein bulls in the HF level contained proteins of about 33.71 kDa that were not detected in the LF. Conclusion: The sperm protein with a molecular weight of 33.71 kDa was predicted to be a specific protein biomarker that influences bull fertility. Sperm fertilization abilities were also determined by the sperm proteins, the morphology of sperm acrosomes, and the quality of plasma membranes. This method can be used to select bulls with high fertility to increase the population of Holstein bulls.

P–025 Sperm selection using a modified “swim up” technique in absence of sperm centrifugation improve sperm DNA fragmentation and decreases miscarriage rate

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Cabell. Vives ◽  
P Belchin ◽  
C Lopez-Fernandez ◽  
M Fernandez-Rubio ◽  
J Guerrero-Sanchez ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Sperm Quality ◽  
P Value ◽  
Sperm Dna Fragmentation ◽  
Sperm Selection ◽  
Male Factor ◽  
Miscarriage Rate ◽  
Sperm Dna ◽  
Group 2

Abstract Study question Is it useful to avoid sperm centrifugation in laboratory routine work to improve sperm quality and reproductive outcome in Assisted Reproduction Techniques (ART)? Summary answer Exclusion of sperm centrifugation for sperm selection using neat sperm samples (IO-lix), increases sperm quality in the collected subpopulation decreasing miscarriage rate after using ICSI. What is known already Inclusion of sperm centrifugation in ART is an aggressive intervention for sperm selection with ineludible production iatrogenic damage affecting sperm integrity. The application of IMSI, PICSI or microfluidic devices avoid sperm centrifugation and may improve the quality of the subsample obtained. However, these methodologies may result time consuming, expensive or producing poor results when the quality of the sperm is limited. We have already shown that a modified swim-up avoiding centrifugation (called IO-lix) is a low-cost and efficient alternative to microfluidic devices, recovers 100 times more concentration and reduces sperm DNA fragmentation with no significant differences to other methodologies. Study design, size, duration This is a retrospective study from 2018 to 2020 which includes patients with an average of age of 38.2 years using their own oocytes with ICSI as fertilization technique. Two aleatory groups of patients were made: Group 1: 88 cycles with 503 fertilized oocytes and 206 blastocysts were obtained with sperm samples processed by IO-lix and Group 2: 303 cycles, 1451 fertilized oocytes and 591 blastocysts using a standard “swim up” technique to process sperm. Participants/materials, setting, methods A total of 391 ICSI cycles were included in this retrospective study. The male factor was similar in both groups and they showed altered SDF previously to the cycle. We compared data of the motility and SDF of sperm samples before and after applying IO-lix and we analyzed by X2 contingence test differences on miscarriage rates between groups 1 and 2. Main results and the role of chance General sperm parameter changes after IO-lix showed that averaged sperm concentration observed in neat ejaculated samples was 62M/SD=46.4. Values obtained after IO-lix in the same samples were 12.3M/SD8.0. Averaged sperm motility in neat samples was 54%/SD=9.3 and 70.9%/SD=13.2 after IO-lix. Finally, sperm DNA fragmentation in neat samples was 35.8%/SD17.3, while these values decreased to 9.2%/SD=3.9 after IO-lix. About reproductive outcome results, significant differences were not obtained on the development to blastocyst stage rate comparing both groups (X2=0.003; p value = 0.954; Alpha 0.05). In the case of IO-lix processed samples, the pregnancy rate was 59.42% in Group 1 and 44.72% in Group 2 (X2=0.651; p value =0.419; Alpha 0.05). A total of 9 miscarriages of 41 clinical pregnancies (21.95%) were observed after IO-lix, while this number increases to 59 out of 123 clinical pregnancies, which means the 47.96% of the embryo transfers, when “swim-up” was used. In this case significant differences were obtained (X2=3.935; p value = 0.0.047; Alpha 0.05). Limitations, reasons for caution Being a pilot study aimed to understand the results of IO-lix in ART, correlations have not been stablished between the levels of sperm improvement after IO-lix and paired results of ART. This study would be necessary, specially to identify the possible origin of miscarriage associated to the male factor. Wider implications of the findings: Elimination of sperm centrifugation using a combined strategy of gradients and “swim-up” for sperm isolation, reduce miscarriage rate and produce equivalent results of blastocyst development to those obtained with “swim-up”. Being a cost-effective and improving laboratory workload, its use for sperm selection is recommended. Trial registration number Not applicable

scholarly journals Improvement of Post-Thaw Sperm Kinematics and DNA Integrity of Cross-Bred Bovine Sperm by Incorporating DGC as Selection Method Prior to Cryopreservation

2017 ◽  
Vol 9 (13) ◽  
pp. 24
Author(s):  
Nur Hilwani Ismail ◽  
Khairul Osman ◽  
Farida Zuraina Mohd Yusof ◽  
Syarifah Faezah Syed Mohamad ◽  
Farah Hanan Fatihah Jaafar ◽  
...  
Keyword(s):  
Treatment Group ◽  
Sperm Quality ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Dna Integrity ◽  
Sperm Selection ◽  
Sperm Analysis ◽  
Fragmentation Index ◽  
Bovine Spermatozoa ◽  
Kinematics Parameters

The aim of this study was to assess post-thaw sperm quality following initial sperm selection using density gradient centrifugation (DGC) prior to cryopreservation. Ejaculates from four mature Charolais cross Kedah-Kelantan bulls were collected using artificial vagina at IBVK Pahang, Malaysia. The ejaculates were aliquoted into 3 groups: non-cryopreserved group (NC); control group of cryopreserved sperm without DGC (ND) and treatment group of sperm undergoing DGC sperm selection before cryopreservation (CDGC). Prior to analysis, samples from both cryopreserved groups were thawed at 37 °C for 30 sec. All samples were analysed for kinematics parameters, viability and compromise in DNA integrity (evaluated as DNA Fragmentation Index, DFI). All kinematics parameters were analysed using computer aided sperm analysis (CASA). Results indicated significant (p < 0.05) kinematics parameter changes for all parameters of velocity (VCL, VSL, VAP) and progression (WOB, LIN, ALH and BCF). Unfortunately, changes in spermatozoa straightness were insignificant (STR) F(2, 68) = 1.004, p = 0.371. Spermatozoa viability had increased by 26.2% (p < 0.01) following the treatment. DFI revealed the treatment group recorded a significant reduction in DFI value (0.17% fragmented DNA). In conclusion, DGC sperm selection prior to cryopreservation reduced the effects of cryodamage and showed an improvement in post-thaw sperm quality, thus reducing the occurrence of asthenozoospermia in populations of frozen-thawed cross-bred bovine spermatozoa.

3 SELECTION OF UNFRAGMENTED-DNA SPERMATOZOA FROM HEAT STRESSED MICE BY FEMALE UTERINE TRACT AND ZONA PELUCIDA BINDING

2009 ◽  
Vol 21 (1) ◽  
pp. 102
Author(s):  
J. D. Hourcade ◽  
M. Perez-Crespo ◽  
B. Pintado ◽  
A. Gutiérrez-Adán
Keyword(s):  
Heat Treatment ◽  
Dna Damage ◽  
Sperm Motility ◽  
Tail Length ◽  
Dna Integrity ◽  
Sperm Selection ◽  
Epididymal Sperm ◽  
Cleavage Rate ◽  
Sperm Dna ◽  
Selection Of

Physiological bases of the sperm selection processes within the female reproductive tract before they meet and fertilize the oocyte are unknown. The aim of this work was to determine if one of the keys of spermatozoa selection could be DNA integrity. It has been reported that sperm DNA damage does not impair in vitro fertilization (IVF). However, it has been suggested that the zona pelucida (ZP) is able to select spermatozoa with unfragmented DNA (Liu and Baker 2007 Hum. Reprod. 22, 1597–1602). In this work, DNA damage of spermatozoa was artificially induced by scrotal heat treatment (HT) (42°C, 30 min). Twenty-one days after the HT, spermatozoa were recovered from the epididymis caudae of CD1 mice and from the uterine horns near the cervix (Uc), from the uterine horns near the oviducts (Uo), and from the oviducts (Ov) of CD1 females 1–2 h after mating with HT and control males. In each region we determined numbers of spermatozoa, individual motility and sperm DNA integrity by COMET assay (% DNA in tail, tail length, and COMET moment was calculated). Also, females naturally mated either with HT or control males were killed at Day 14 of pregnancy, and number of foetuses and resorptions was recorded. Additionally, IVF was performed with epididymal sperm from HT or control males, Two hours after IVF attached and un-attached spermatozoa to the ZP were recovered and samples were evaluated for sperm motility (CASA), sperm zona-binding, and sperm DNA fragmentation (COMET). Also cleavage rate of fertilized oocytes with sperm from HT or control males was analyzed. One-way ANOVA was used to compare the results form each group. Epididymal sperm count (12*106 and 4.4*106 for control and HT respectively), sperm motility (75 and 21% respectively) and testis weight (133.90 and 68.76 mg, respectively) were significantly reduced after heat treatment (P < 0.001). For the heat treatment, COMET values decreased significantly during the transit from Uc to Uo and from Uo to Ov (Tail DNA: 25.7, 23.5, and 14.4% respectively, P < 0.01; Tail length: 38.4, 29.4, and 11.2 pixels, P < 0.001; COMET Moment: 12.5, 8.5, and 2 respectively, P < 0.001). Heat treatment reduced numbers of foetuses (7 ± 0.5 v. 5 ± 0.49, control and HT group, respectively), but number of resorptions was not altered. Spermatozoa bound per ZP in IVF experiments (55 ± 7 and 13 ± 6, control and HT, respectively) and cleavage rate (61 ± 1 v. 15 ± 6, control and HT, respectively) were significantly reduced in the HT group. Two hours after IVF, spermatozoa attached to the ZP in HT group showed a significant decrease in COMET parameters as in tail length (59.46 ± 2.895 v. 34.66 ± 3.531), and in tail moment compared with unattached spermatozoa. Our results indicate that DNA integrity sperm selection mechanisms are present in both the female tract and the ZP. We suggest that genital tract and sperm-ZP binding process plays an important role in selection of sperm with normal chromatin DNA.

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