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Apoptosis-like changes in bull sperm and their effects on fertility



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The overall objective of this thesis was to evaluate the effects of apoptosis-like membrane and DNA changes in bull sperm, and to relate these changes to a bull’s fertility potential. This thesis hypothesis is that apoptosis-like changes occurring in fresh or cryopreserved bull sperm have a negative effect on a bull’s fertility potential. Two studies were conducted, the objectives of study 1 were to confirm the relationship of apoptosis-related membrane and nuclear changes in bull sperm with fertility, to predict the fertility of beef bulls used for natural mating; and to evaluate the effect of sperm with nicked-DNA on cleavage and blastocyst formation in vitro. In Experiment 1, phosphatidylserine (PS) translocation, from the inner to the outer plasma membrane, and DNA nicks in the sperm from 50 dairy bulls were determined using Annexin-V/PI and TUNEL assays, respectively. Relationships between the parameters of the assays and the known fertility levels of the bulls were calculated. In Experiment 2, fertility levels of 15 beef bulls used for natural mating were estimated using a regression model of DNA nicks developed in Experiment 1. In Experiment 3, the effect of DNA nicked sperm on cleavage and blastocyst rates were evaluated in in vitro produced embryos, using high and low sperm concentrations (30,000 and 300,000 sperm per IVF droplet) to fertilize the mature oocytes. In Experiment 1, there were significant relationships of fertility with live sperm (P<0.05) and necrotic sperm (P<0.01) (Annexin-V/PI assay), and with DNA-nicked sperm (P<0.001) (TUNEL assay). In Experiment 2, the fertility level of bulls used for natural breeding was estimated and ranged from -7.3 to 2.4. In Experiment 3, the cleavage rate was significantly affected by the number of sperm with nicked DNA, regardless of sperm concentration. At the low sperm concentration, blastocyst rate was significantly lower when higher DNA nicked sperm were used (51% vs 32%; high vs low DNA nicks) (P<0.05). Blastocyst rate was non significant at the higher sperm concentration regardless of DNA nicks. The second objective of this study was to evaluate the effect of apoptosis inhibitors added to post-thaw sperm samples on their longevity, to increase the availability of viable sperm to oocytes for fertilization. Frozen semen from seven bulls was used; six straws from each bull were pooled. Samples included, untreated control (sperm remaining in extender), treated control (washed sperm), and four treatments (inhibitors) each at two concentrations. Apoptosis inhibitors assessed included; Bax channel blocker, z-VAD-FMK, Coenzyme Q10, and XIAP. Motility related characteristics were evaluated using computer assisted sperm analysis (CASA). Membrane intactness and normal acrosomes were evaluated using fluorescein isothiocyanate-peanut agglutination (FITC-PNA)/propidium iodide (PI) assay. Mitochondrial membrane potential was evaluated using Mitotracker Deep Red (MtDR). Sperm parameters were evaluated at 0, 3, 6, and 12 hours of incubation. Our results showed, no significant effect of apoptosis inhibitors on post-thaw sperm motility and structural characteristics. The decline in sperm motility and structural characteristics at 6 h of incubation was lower (P<0.05) in treated control and treatment groups than untreated control group. In conclusion, the presence of nicked DNA in sperm may be used as an estimate of the fertility level of a breeding bull. The levels of sperm with DNA nicks have a negative effect on cleavage rates and subsequent blastocyst development. The second conclusion indicates that the addition of an apoptosis inhibitor post-thaw to semen samples does not improve longevity or fitness, in any of the parameters evaluated. The simple removal of extender showed to be beneficial to sperm longevity and fitness. Further studies are needed to evaluate the cleavage and blastocyst rate of embryos fertilized with a single sperm known to carry DNA nicks. As well, the effect of the addition of apoptosis inhibitors before cryopreservation of bull semen needs to be evaluated.



Apoptosis, Sperm, Bull Fertility,



Master of Science (M.Sc.)


Veterinary Biomedical Sciences


Veterinary Biomedical Sciences


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