Doctor of Philosophy (PhD)


Animal Science (Animal, Dairy, and Poultry Sciences)

Document Type



This study investigated the effects of semen extender constituents and cryopreservation on bovine spermatozoal mitochondrial function. Three yearling Holstein bulls were used. Two ejaculates per bull were collected and pooled on a weekly basis for five weeks and extended in four treatments: 1) sodium citrate egg yolk extender with antibiotics (lincomycin, spectinomycin, gentamicin and tylosin); 2) ¡°1¡± with glycerol; 3) ¡°2¡± without antibiotics; and 4) ¡°1¡± without antibiotics. Each was divided into portions for analyses before freezing and after cryopreservation. The pre-freeze and thawed semen were transferred to a 37¡ãC water bath, the same assays were performed. In experiment 1, resazurin reduction (RD) was measured spectrophotometrically at sequential 25 minute intervals for 125 minutes. In experiment 2, specific activities of cytochrome c oxidase (CytoCox) and citrate synthase (CS) were measured spectrophotometrically immediately post-thaw and after 125 minutes of incubation. In experiment 3, ATP was measured using luciferin-luciferase assay simultaneously with RD. Total and progressive motilities (TM and PM), progressive (PV), curvilinear (VCL) and pathway (VAP) velocities were measured simultaneously with RD, ATP content and CytoCox and CS using computer assisted semen analysis system (CASA). In experiment 4, the NADH dehydrogenase (ND1) gene of mtDNA was sequenced before and after cryopreservation using PCR. Data were analyzed by least square methods; mean differences were delineated by Tukey¡¯s test. In experiment 1, RD differed among treatments (P<0.05). Cryopreservation decreased (P<0.05) RD, TM, PM, PV, VAP and VCL. Resazurin reduction correlated with PM (r=0.45, P<0.05) and TM (r=0.2, P<0.05). In experiment 2, incubation time and incubation with Triton X100 were sources of variation in CytoCox and CS specific activities (P>0.05). Only CS from spermatozoa incubated with Triton X100 correlated with RD (r=0.22, P<0.05). CytoCox and CS did not correlate with motility parameters. In experiment 3, spermatozoal ATP was not different (P>0.05) among treatments. However, cryopreservation decreased (P<0.05) ATP. Spermatozoal ATP correlated with motility parameters (r¡Ý0.65) and RD (r=-0.30) (P<0.05). In experiment 4, the frequency of amino acid change was higher (P<0.05) post-thaw in the treatment containing only antibiotics. Cryopreservation, more than extender constituents impacted mitochondrial function of bovine spermatozoa.



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Committee Chair

John Chandler