2018: SEASONALITY AFFECTS SEMEN CRYOPRESERVATION OF WHITE-TAILED DEER BUCKS (ODOCOILEUS VIRGINIANUS) THROUGHOUT RUT


Seasonality affects semen cryopreservation of white-tailed deer bucks (Odocoileus virginianus) 
throughout rut 

Jamie L. Stewart,a Clifford F. Shipley,a Robyn E. Ellerbrock,ab Fabio S. Lima,a Igor F. Canissoa 
aCollege of Veterinary Medicine, University of Illinois, Urbana, IL; bCollege of Veterinary Medicine, 

University of Georgia, Athens, GA 
 

White-tailed deer farming is a growing industry in the United States, with breeding operations 
contributing significantly to the industry’s economic impact. Artificial insemination with frozen semen 
allows for selection and dissemination of valuable genetics, yet surprisingly little information is available 
regarding the best time throughout rut to perform semen cryopreservation. The objective of this study was 
to compare the efficacy of semen cryopreservation of white-tailed deer bucks collected early in the 
breeding season (September), at peak rut (December), and late season (March). We hypothesized that 
semen freezing ability would be enhanced at peak rut. Mature bucks (n = 7-11) were anesthetized with 
tiletamine-zolazepam (0.88 mg/kg) and xylazine (2.2 mg/kg) administered intramuscularly via projector. 
The penis was manually exteriorized, and semen was collected by electroejaculation (Pulsator IV, Lane 
Manufacturing, Inc). An aliquot of the ejaculate was diluted 1:60 in warm (37°C) Optixcell extender 
(IMV Technologies) and placed into a pre-warmed 20 µM chamber slides (Vitrolife, Microcell Counting 
Chambers) for assessment of motility via computer-automated sperm analysis (Spermvision II, MiniTube 
of America). The remainder of each sample was extended with Optixcell to a final concentration of 120 
million sperm/mL. Ejaculates were cooled to 5°C over 1 h, loaded into 0.5-mL straws, and incubated for 
3 to 5 h. Straws were frozen manually in liquid nitrogen vapors at 4 cm above the liquid nitrogen level for 
15 m before submerging them for final freezing and storage. Each semen straw was thawed in a 37°C 
water bath for 30 s for post-thaw analyses. Overall and progressive sperm motilities were evaluated in 
each sample using computer-automated semen analysis. Additionally, samples were stained with 
fluorescent probes and subjected to flow cytometry for characterization of sperm viability (SYBR-14/PI), 
acrosome integrity (FITC-PNA/PI), and chromatin stability (acridine orange). Data were analyzed using a 
Kruskal-Wallis chi-squared test with a Dunn posthoc test (non-parametric) or ANOVA with repeated 
measures and Tukey post-hoc test (parametric) in R Version 3.2.2 (https://www.r-project.org/). Pre-freeze 
overall and progressive sperm motilities were lowest in March and highest in December (p≤0.04). 
However, post-thaw overall and progressive motilities were lowest in September and highest in December 
(p=0.01). The DNA Fragmentation Index was lowest in December (p<0.01), but did not differ between 
March and September (p=0.23). The percentage of live sperm cells was highest in December (p=0.03), 
and the percentage of intact acrosomes per live sperm cell was highest in September (p=0.02). This study 
confirms that bucks have superior semen quality when in peak rut (December). Though semen collected 
early or late in rut may present acceptable motility, DNA stability appears to be impaired, which could 
adversely affect fertility rates. Therefore, we suggest that semen cryopreservation be performed closer to 
peak rut to optimize post-thaw semen quality and ensure successful artificial insemination outcomes. 

 
Keywords: Andrology, cervid, fluorescent probes, semen cryopreservation 

Clinical Theriogenology • Volume 10,  Number 3 • September 2018 304