Interrelationship Between Plasma Membrane Integrity, Mitochondrial Membrane Potential and DNA Fragmentation in Cryopreserved Bovine Spermatozoa

Abstract

The objective of this flow cytometric study was to examine plasma membrane integrity, mitochondrial membrane potential (MMP) and the degree of DNA fragmentation of cryopreserved bovine sperm immediately (0 h) and 3 h after thawing and to compare the results with each other and with the fertility of bulls. Cryopreserved spermatozoa from 4 consecutive ejaculates of 20 bulls were examined. Percentages of plasma membrane intact sperm (PMI) and sperm showing a high MMP (HMMP), respectively, were determined by the SYBR14/PI- and the JC-1 assays. DNA fragmentation was analysed by the standard deviation of the DNA fragmentation index (SD-DFI) and the percentage of sperm with a high degree of DNA fragmentation (%DFI) by using SCSA(TM). The mean non-return rate on day 56 (NRR 56) ranged from 63.7% to 78.0% (mean +/- SD: 71.8% +/- 3.7%). Mean values for PMI and HMMP decreased from 37.4% +/- 6.8% to 31.2% +/- 6.1% and from 38.8% +/- 7.1% to 23.8% +/- 7.7% respectively. SD-DFI increased from 56.9% +/- 8.0% to 69.0% +/- 12.9% and %DFI from 6.4% +/- 2.5% to 12.4% +/- 5.8%. The correlation between PMI 0 h and HMMP 0 h (r = 0.95; p < 0.0001) was higher (p < 0.05) than that between PMI 3 h and HMMP 3 h (r = 0.88; p < 0.0001). %DFI 0 h was neither related to PMI 0 h nor to HMMP 0 h (p > 0.05), nor was there a correlation (p > 0.05) between DFI 3 h and PMI 3 h; but %DFI 3 h and HMMP 3 h were significantly correlated (r = -0.31; p < 0.05). SD-DFI and %DFI 3 h were the only parameters related to NRR 56 (r = -0.58; p < 0.05). In conclusion, plasma membranes and mitochondria are similarly affected by the freezing and thawing process, but not during the incubation period after thawing.

Evaluation of bull semen for fertility-associated protein,in vitrocharacters and fertility

“…Evenson (1999) suggested that loss of DNA integrity ≥ 20% might announce lower fertility. Loss of DNA integrity during incubation in the present study was less than in the reports of Bollwein et al (2008) who reported the per cent of DNA fragmentation index increased from 5.70 ± 1.50 at immediate post thaw to 10.90 ± 4.40 at 3 h of incubation. In the present study, group I bulls that were positive for HBP had significantly more number of DNA intact cells than the group II bulls during incubation.…”

“…In vitro sperm characters were evaluated at immediate post thaw, 60, 120 and 180min post-thaw incubation (Bollwein et al 2008). Motility analysis was conducted using a computer-assisted semen analyser (Sperm Class Analyzer, Microptic, Barcelona, Spain) as described by Selvaraju et al (2008).…”

Reduced glutathione and cysteine hydrochloride on crossbred bull semen

“…Mean values for PMI (plasma membrane intact sperm) and HMMP (high 31 mitochondrial membrane potential) decreased from 37.4% +/-6.8% to 31.2% +/-6.1% and from 38.8% +/-7.1% to 23.8% +/-7.7% respectively. SD-DFI (standard deviation of the DNA fragmentation index) increased from 56.9% +/-8.0% to 69.0% +/-12.9% and %DFI from 6.4% +/-2.5% to 12.4% +/-5.8% (Bollwein et al, 2008).…”

“…DNA fragmentation index in Control , 1 mM GSH and 5 mM GSH were 1.2 ± 0.1, 0.8 ± 0.1 and 0.8 ± 0.2 respectively (Joaquı et al, 2007) Bollwein et al (2008 analyzed DNA fragmentation by the standard deviation of the DNA fragmentation index (SD-DFI) and the percentage of sperm with a high degree of DNA fragmentation (%DFI) by using SCSA (TM) .…”

Efecto de la manipulación del semen criopreservado de bovinos Bos Taurus sobre la integridad espermática

“…Los resultados en la evaluación de la IMp y de la IMa son muy relevantes debido a que el ORT ha demostrado tener una correlación alta y positiva con la capacidad fecundante del espermatozoide (38). Sin duda alguna, las fallas en la manipulación de la pajilla afectan la funcionalidad espermática causando un efecto deletéreo sobre la función del acrosoma (39, 40) y de la membrana plasmática, potencial de membrana mitocondrial e integridad del ADN (41). Los espermatozoides sometidos a injuria están sujetos a una serie de alteraciones en su estructura, tales como dilatación y rompimiento a nivel de la membrana, con cambios de permeabilidad en esta y cambios en la estructura de fosfolípidos y proteínas, que traen como consecuencia una reducción en su motilidad y viabilidad espermáticas (42,43).…”