Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

2018 ◽  
Vol 54 (2) ◽  
pp. 184-194 ◽  
Author(s):  
Camila Arroyo-Salvo ◽  
Francisco Sanhueza ◽  
Fernanda Fuentes ◽  
Favián Treulén ◽  
María Elena Arias ◽  
...  
Keyword(s):  
Sperm Capacitation ◽  
Fluid Medium ◽  
Human Tubal Fluid

156 Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

2019 ◽  
Vol 31 (1) ◽  
pp. 203
Author(s):  
R. Felmer ◽  
C. Arroyo-Salvo ◽  
F. Fuentes ◽  
P. Cabrera ◽  
F. Treulen ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Tyrosine Phosphorylation ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Sperm Quality ◽  
Membrane Damage ◽  
Sperm Capacitation ◽  
Fluid Medium ◽  
Motility Parameters ◽  
Human Tubal Fluid

Conventional IVF has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. Thus, the first part of this study aimed to compare HTF (Summers and Biggers, 2003 Hum. Reprod. Update9, 557-582, DOI: 10.1093/humupd/dmg039) and Whitten’s (McPartlin et al. 2009 Biol. Reprod.81, 199-206, DOI: 10.1095/biolreprod.108.074880) media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine on sperm motility parameters was evaluated in both media at different incubation times. Fresh semen from 3 Chilote stallions was collected, diluted to 10×106 sperm mL−1 in capacitating (7mg mL−1 BSA and 25mM NaHCO3) and non-capacitating (without BSA and NaHCO3) HTF and Whitten’s media and incubated for 30 and 120min at 38°C in air atmosphere. Integrity and destabilisation of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (ΔΨm) using tetramethylrhodamine methyl ester perchlorate, acrosome membrane integrity by peanut agglutinin/fluorescein isothiocyanate and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® in a FACSCanto II flow cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ, USA). A total of 10,000 sperm events were acquired from each measurement (n=3 replicates for each stallion). Motility parameters were evaluated using the integrated semen analysis system (ISAS®, Selinion Medical, Brussels, Belgium). Percentage data were arcsine transformed and subjected to a 2-way ANOVA with Bonferroni’s post hoc test using Prism 7 software (GraphPad Software, La Jolla, CA, USA). We found no differences between Whitten’s and HTF media in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30 and 120min of incubation. Membrane fluidity (MC540) increased in both media at 30 and 120min of incubation compared with non-capacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2 and 4h of incubation compared with non-capacitating conditions, without differences between media. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm. Funding support was received from FONDECYT 1160467 CONICYT, Chile.

scholarly journals R-075. Human embryo development in a modified human tubal fluid medium (P1 medium preimplantation stage one) lacking glucose and phosphate

1999 ◽  
Vol 14 (Suppl_3) ◽  
pp. 312-313
Author(s):  
R. Fabbri ◽  
P.M. Ciotti ◽  
O. Magrini ◽  
T. Marsella ◽  
C. Lagalla ◽  
...  
Keyword(s):  
Embryo Development ◽  
Human Embryo ◽  
Fluid Medium ◽  
Preimplantation Stage ◽  
Human Embryo Development ◽  
Human Tubal Fluid

202 SEX PRE-SELECTION IN RABBITS: AN ATTEMPT TO SKEW OFFSPRING SEX THROUGH PERCOLL AND SWIM-UP SPERM PREPARATION TECHNIQUES

2012 ◽  
Vol 24 (1) ◽  
pp. 213
Author(s):  
M. Copello ◽  
A. Perez ◽  
S. Marquez ◽  
M. Sansinena
Keyword(s):  
Body Condition Score ◽  
Sperm Concentration ◽  
Female Offspring ◽  
Sample Volume ◽  
Fluid Medium ◽  
Sperm Preparation ◽  
Female Birth ◽  
Underlying Mechanisms ◽  
Preparation Techniques ◽  
Human Tubal Fluid

Sperm preparation techniques could have an effect on the birth ratio (male vs female offspring) through enrichment of either X- or Y-bearing sperm populations, although studies have been poorly controlled and results have been inconclusive. In the rabbit, producers may be interested in producing a majority of males (meat producing systems) or females (hybrid producing systems). The objective of this study was to evaluate the effect of modified Percoll and Swim-up protocols on the enrichment of the male and female birth ratios in rabbits. Four hybrid mature bucks of adequate body condition score and proven fertility were used. The same four bucks were used throughout the study (8 replicates). Ejaculates were collected using a warmed, solid, artificial vagina. Progressive motility was assessed at 200× magnification under brightfield microscopy on a heated (37°C) stage. Sperm concentration for each buck was determined using a Neubaur chamber and adjusted (350 × 106 total spermatozoa mL–1); they were then combined (1 mL/buck) to form a heterospermic sample (final heteropsermic sample volume = 4 mL). The heteropsermic sample was then divided into 3 sperm preparation treatments: (1) diluted in commercial tri-buffered extender followed by immediate AI (control); (2) modified Percoll centrifugation in a 90 to 45% density gradient (diluted in human tubal fluid medium); and (3) Swim-up (in human tubal fluid medium). In total, 125 females were inseminated. Does were treated with PMSG (20 UI/doe, 48 h before AI) and inseminated with 30 to 40 × 106 spermatozoa; ovulation was induced with 20 μg of gonadotropin-releasing hormone (GnRH)/doe immediately after AI. Sex of newborn kits was determined 35 days after birth by an experienced technician through external visualization of genitalia. Results were statistically analyzed using ANOVA and Tukey test (Infostat). The insemination of does with heterospermic ejaculates (control) resulted in a progeny distribution of 49 ± 2% males and 51 ± 2% females, which is in good agreement with the expected theoretical 50:50 ratio. Sex ratios from modified Percoll and Swim-up treatments deviated significantly from the control (P < 0.01). Percoll gradient resulted in a progeny distribution of 32 ± 2% males and 68 ± 2% females; Swim-up resulted in a 64 ± 2% male and a 36 ± 2% female distribution (P < 0.01), whereas prolificacy, perinatal mortality and birth weight were not affected by treatment (Table 1). Our results indicate sperm preparation techniques could be used to skew birth ratios towards male or female offspring in rabbits; Percoll preparation could be used to increase proportion of female kits whereas Swim-up could be used to increase the proportion of males. Further studies are needed to determine the underlying mechanisms of action. Table 1.Progeny distribution after Percoll or Swim-up sperm preparation in rabbits

Medium effects on capacitation and sperm penetration through the zona pellucida in inbred BALB/c spermatozoa

Zygote ◽  
2005 ◽  
Vol 13 (2) ◽  
pp. 145-153 ◽  
Author(s):  
Seiji Kito ◽  
Yuki Ohta
Keyword(s):  
Zona Pellucida ◽  
Inbred Strains ◽  
Control Medium ◽  
Sperm Capacitation ◽  
Medium Effects ◽  
Sperm Penetration ◽  
Pronuclear Formation ◽  
Human Tubal Fluid ◽  
Poor Fertilization

Inbred BALB/c mice are one of the most difficult inbred strains to fertilize in vitro. In this study we examined the abilities of various media used for mouse in vitro fertilization (IVF) to support capacitation and sperm penetration through the zona pellucida (ZP) of inbred BALB/c spermatozoa. Media examined were TYH, M16, CZB, mWhitten medium, T6, modified Tyrode's solution (mTyrode's), mKSOM, MEM and TCM199. Modified human tubal fluid (mHTF) was used as a control medium. When sperm were capacitated and inseminated in the same medium, mHTF showed the best fertilization (approximately 80%) scored by male pronuclear formation (<26%) at 5 h post-insemination (PI). When sperm were capacitated in various media and inseminated in mHTF, sperm capacitated in TYH solution (93%) but no other media (<45%) showed a significantly higher level of sperm nuclear decondensation (SND) than mHTF at 2 h PI (approximately 65%). When sperm were capacitated in mHTF and inseminated in various media, only mTyrode's (52%) was not significantly lower than mHTF (66%) in terms of SND at 2 h PI (<49%). Sperm capacitation also was examined by chlortetracycline (CTC) staining. Sperm capacitated in TYH solution showed a significantly higher percentage of capacitation (46%) than those treated in HTF (28%) and other media (<24%). These results indicate that the best approach for IVF in the BALB/c strain is capacitation in TYH and insemination in mHTF. Poor fertilization of BALB/c may result from suboptimal conditions of sperm capacitation and insemination, and overall IVF success may differ depending on strains used.

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