Animal Reproduction Science 184 (2017) 196203

Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Assessment of butorphanol-azaperone-medetomidine combination MARK

as anesthesia for semen collection and evaluation of semen quality
in white-tailed deer (Odocoileus virginianus)

S.M. Kirschnera, , R. Rodenkirchb
a
SR Veterinary Technologies, R & D Department,1230 West Ash Street, Windsor, CO, USA
b
Rodenkirch Whitetails & Genetics, Research Lab,867 Barbara Street, Sun Prairie, WI, USA

AR TI CLE I NF O AB S T R A CT

Keywords: The aim of this current study was to evaluate the level of anesthesia produced by a combination
Anesthesia of butorphanol-azaperone-medetomidine (BAM) for semen collection by electroejaculation on
Azaperone captive white-tailed bucks (Odocoileus virginianus). Ten male white-tailed deer, weighing
BAM 68.2115.9 kg, ranging in age from one to four years were randomly selected from housing pens
Butorphanol
and anesthetized with the BAM drug combination at a dose volume of 2.0 mL each. Semen was
Medetomidine
Semen collection
collected from each animal using a standard cervid electroejaculation protocol while under BAM
White-tailed deer anesthesia. Physiological data was recorded following induction of anesthesia and during semen
collection. Collected ejaculates were prepared for analysis using a standard extender protocol for
cryopreservation. Eleven sperm viability parameters were quantied for each sample using a
Computerized Assisted Sperm Analysis system, including total seminal volume; sperm con-
centration and total sperm number. kinematic parameters of motile spermatozoa were also as-
sessed. Results demonstrated that BAM provided an eective plane of anesthesia for successful
collection of viable sperm. Measured physiological variables of heart rate, respiration and body
temperature all remained within safe, normal limits. Data recorded on semen characteristics from
all collected ejaculates correlated well with key traits determined to be important for successful
fertilization through measurement of total semen volume; sperm concentration; total sperm
number; and kinematic parameters of motile spermatozoa. There were no serious adverse events.
This eld study indicates that BAM anesthesia is suitable for semen collection in white-tailed
deer.

1. Introduction

Electroejaculation (EE) is the prevalent method of sperm collection in cervids and is used to repeatedly recover semen samples
from selected males. (Garde et al., 2006) Historically the most common anesthesia protocol utilized for this procedure has been the
drug combination of tiletamine/zolazepam and xylazine hydrochloride administered intramuscularly. (Monteith et al., 2012) This
combination has been used in deer for over 30 years. Tolazoline hydrochloride or atipamezole hydrochloride is generally used to
reverse this anesthesia after semen collection. Tiletamine/zolazepam and xylazine hydrochloride anesthesia has been known to
induce such adverse side eects as acidemia, bloating, regurgitation, bradycardia, hyperthermia, hypoxemia, glycosuria, and an-
orexia. (Miller et al., 2009)

Corresponding author at: 1230 West Ash Street, Windsor, Colorado, USA.
E-mail addresses: skirschner2@gmail.com, skirschner@srvet.net (S.M. Kirschner).

http://dx.doi.org/10.1016/j.anireprosci.2017.07.016
Received 21 April 2017; Received in revised form 12 July 2017; Accepted 24 July 2017
Available online 25 July 2017
0378-4320/ 2017 Elsevier B.V. All rights reserved.
S.M. Kirschner, R. Rodenkirch Animal Reproduction Science 184 (2017) 196203

Recently, a novel drug combination of butorphanol-azaperone-medetomidine has been developed for use as an anesthetic in non-
domestic species. The site-specic alpha-2 adrenergic agonist medetomidine has been administered as an alternative to xylazine for
veterinary use for many years. It has proven to be a reliable, more potent alpha-2 agonist that can be quickly reversed by the alpha-2
antagonist atipamezole. Published studies combining medetomidine with butorphanol, a morphine-based schedule IV opioid, have
reported eective immobilization of deer based on its synergistic central nervous system sedation (Miller et al., 2009). With addition
of the neuroleptic sedative azaperone to this butorphanol/medetomidine combination, the formulation is abbreviated as the acronym
BAM.
Since its introduction several published studies evaluating the ecacy of BAM have reported safe, rapid inductions and eective
planes of anesthesia in several species (Miller et al., 2009; Siegal-Willott et al., 2009). These studies have also indicated a smoother,
more rapid recovery when reversed with atipamezole and naltrexone antagonists versus traditional xylazine anesthetic combinations
(Miller et al., 2009).
The BAM combination of butorphanol-azaperone-medetomidine administered in this study was a premixed solution containing
27.3 mg of butorphanol tartrate, 9.1 mg of azaperone tartrate, and 10.9 mg of medetomidine hydrochloride per 1.0 mL volume. This
concentration of these pharmaceuticals enables convenient delivery of anesthesia using a single low-volume dart (Lance, 2008).
This study was the rst assessment of semen collected from a single group of test bucks under BAM anesthesia.

2. Materials and methods

2.1. Animals

Ten white-tailed bucks were anesthetized using BAM, with one semen ejaculate per buck collected (on either December 8 or 9,
2015) at a captive breeding facility in Gillett, Wisconsin. All deer were identied by ear tags. Deer body weight ranged from
68.2115.9 kg with age ranging from one to four years. Extensive studies have documented that the rutting period (the time each
year when white-tailed deer are sexually active) typically occurs between October 1 and January 1 in Northern Wisconsin. During
this breeding season, males are characterized by having hard antlers and maximal testis volume, proven to be associated with high
levels of semen volume, sperm concentration, percentage of motile sperm, and serum testosterone concentrations (Mirarchi et al.,
1975).
Division of Wildlife records have chronicled that white-tailed deer breeding begins in late October and peaks between November
3 and November 16 north of latitude 40 (Resources, 2001) Our selected study collection dates correlate positively with this optimal
white-tailed breeding season in Gillett, Wisconsin (latitude 44.89 North).
Test group bucks were housed with individual breeding groups of does in separate multi-acre pens. However, since all mature
does within the captive herd were bred via articial insemination (through laparoscopic AI procedures) conducted November 5 and
6, 2015, males were not introduced into their assigned breeding group until November 20 or 21, 2015. With conrmed AI conception
rates at this test facility documented to average 65% among each breeding group of 920 does, it could be estimated that test bucks
had a limited opportunity to breed through natural cover with approximately 47 receptive does exhibiting estrus behavior.
Published studies on male breeding success among both captive and wild white-tailed deer populations have reported the ability of
individual bucks to successfully breed numerous does throughout the rut period (Ott et al., 2003; Sorin, 2004).
On the Wisconsin captive white-tailed deer facility where this study was performed (housing a herd of approximately 1500 head),
a review of genetic breeding records compiled through use of modern paternity analyses, provided detailed ospringsire re-
lationships conrming their resident bucks have naturally bred up to 30 does in one season. In one published study conducted among
wild white-tailed deer populations, Sorin analyzed genetic paternity assignments within a wild group of 47 females and 17 dierent
males on a game reserve in southeastern Michigan. Tabulated results armed how some individual bucks mated with up to 7 females
during one breeding season (Sorin, 2004). Reports from another study by Ott conrmed that individual male white-tailed deer were
able to breed up to 64% of the females in two 200-ha enclosures populated with wild deer (Ott et al., 2003). Following review of
database records from our captive deer test facility combined with previously-published studies of wild white-tailed deer populations,
provided condence that semen collections from animals in this study would provide acceptable ejaculate volumes and sperm
quality. In addition, semen collections did not begin for a minimum of 30 days following nal attempts for test bucks to naturally
breed females, providing condence that sucient levels of testicular spermatozoa would be present for collection.
It was determined for this study to select bucks continuously housed with mature does based on previous trials reporting how
continuous contact with females had enhanced male reproductive function in several species. While a recent study (Villagrn and
Ungerfeld, 2013) conrmed that female presence within contained breeding groups resulted in increased testosterone levels and
enhanced seminal parameters in pampas deer males (Ozotoceros bezoarticus), additional publications have reported this same cor-
relation in other cervid species and non-domestic ruminants.
In 2004, Martin et al. discussed the socio-sexual signals and resulting stimulatory eects of males on females, of females on males
and of females on females in many species (Martin, 2004). In addition to pampas deer males, this increase in testosterone was also
observed in other cervids of diering social hierarchy including studies in wapiti (Haigh et al., 1984) Eld deer (Monfort et al., 1993)
and red deer (Malo et al., 2009) Beyond cervids, a 1976 study by Illius et al. reported on the extreme social stimulus of housing male
sheep in the presence of cyclic females and the resulting increased plasma testosterone levels in these rams (Illius et al., 1976).

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2.2. Drug dosage and administration

Each study animal was administered BAM using a single, 3.0 cc disposable RDD (remote drug delivery) dart with gel collar and 1
tri-port cannula (Pneu-Dart, Inc., Williamsport, Pennsylvania) delivered into a muscular region of the hind quarter. The BAM for-
mulation (ZooPharm, Laramie, Wyoming, USA) was dosed based on previously published studies (Miller et al., 2009). BAM was
administered to each animal at a volume of 2.0 mL that contained 54.6 mg of butorphanol, 18.2 mg of azaperone, and 21.8 mg of
medetomidine.
While test animal bodyweight ranged from 68.2115.9 kg a 2.0 mL dose volume of the BAM anesthetic was administered to all
bucks. Based on ndings from previously published clinical trials and extensive eld experiences, the recommended 2.0 mL dose
volume rate has been proven to provide a level of ecacious and safe anesthesia when administered to white-tailed bucks of varying
bodyweights (Mich, 2008; Siegal-Willott et al., 2009). In addition to the complete immobilization and excellent induction quality
reported in these cited trials, determination of this optimal recommended dosage for a broad bodyweight range of male white-tailed
deer by the BAM developers also applied specic allometric scaling data (relating metabolic rate and body mass) as presented by
Sedgwick (Sedgwick, 1993). The resulting low volume drug dose was purposefully formulated to enable ecient eld delivery of this
combination anesthesia via a single RDD projectile syringe.

2.3. Semen collection

Semen was collected by electroejaculation as rst described by Martnez-Pastor et al. (Anel-Lopez et al., 2017; Martinez-Pastor
et al., 2009). In order to avoid urine contamination during this study, collection tubes were changed between semen emissions as
previously indicated (Martinez-Pastor et al., 2009).
An auto-adjust Lane Pulsator IV Electronic Ejaculator with a Lane Ram Ejaculator probe (Lane Manufacturing, Denver, CO) was
used for the procedure. The probe with three ventrally located longitudinal brass electrodes was inserted into the rectum. Electrical
stimulations with this probe were delivered in a pulsatile pattern, administering from 2.0 V to 4.0 V pulses (approximately 35 s in
duration). An average of four stimulations were given and when the deer began ejaculating, electrical pulses were continued until
ejaculation ceased. When the procedure appeared ineective on an individual animal, another four stimulations were performed
following a ve-minute break at a slowly increased power setting (not to exceed 4.0 V).
Each ejaculate was collected in a 50 mL plastic tube until the semen sample no longer appeared cloudy (indicating lack of sperm
cell presence). Immediately following collection, the sample ejaculate tube was placed into a warmer (Minitube Plate/Stage Warmer,
Minitube of America, Warner, WI) preset at 37 C, in preparation for quantitative evaluation of multiple sperm viability parameters.

2.4. Physiological parameters measurement

Heart rate, respiration rate, and rectal temperature were measured and recorded beginning at induction of anesthesia during each
collection procedure. Following completion of each collection, and prior to anesthesia reversal, bucks received 1000 mL (IV) of
lactated ringers solution (Abbott Laboratories, IL), containing 3 mL of water-soluble vitamin B complex and 2 mL of unixin me-
glumine (Prevail 50 mg/mL, VetOne, Boise, ID). Animals were also administered 5 mL intramuscular injection of a Vitamin A,
Vitamin D3, and Vitamin E solution (Vitamin E-AD Injection, VetOne, Boise, ID).

2.5. Reversal of BAM agonists

Each deer was returned to its assigned holding pen while still under anesthesia and appropriate antagonist were administered to
reverse the BAM anesthesia. Reversal drugs were injected intramuscularly (IM) as follows: 100 mg atipamezole HCl at 25 mg mL-1;
(ZooPharm, Laramie, WY) to reverse the eects of medetomidine and 25 mg naltrexone HCl at 50 mg mL-1 (ZooPharm, Laramie, WY)
antagonizing the butorphanol sedation.
Recovery time was recorded as the time elapsed from injection of atipamezole and naltrexone to when there were no longer signs
of sedation and animal behavior appeared normal. Quality of recovery was quantied using previously established cervid behavioral
criteria and evaluated on a scale from 5 to 0 (Miller et al., 2009). Recovery from sedation scores were assigned as follows: 5 = lateral
recumbency with no sign of reversal; 4 = lateral recumbency, unable to maintain an erect head, and noticeable eye or ear movement;
3 = unable to stand, dazed and unsteady, but able to hold head up; 2 = standing with moderate ataxia, braced stance, and some-
times lowered head; 1 = minimal sedation characterized by drooping eyelids; and 0 = no sign of sedation (Miller et al., 2009).

2.6. Viability assessment of collected semen

From each collected ejaculate sample, 2.0 L of semen was placed into a separate aliquot tube with the addition of 200.0 L of
TRIS-based semen extender (BoviPRO CryoGuard, Minitube, WI), pre-warmed to 37 C on the plate/stage warmer. A 5.2 L sample
was drawn from this 1:100 dilution ratio semen-extender aliquot and loaded into a counting chamber (Leja 20 m-4 Chamber Slide,
Leja Intl, The Netherlands). The lled counting chamber slide was then placed on the Computerized Assisted Sperm Analysis (CASA)
heated microscope stage set to 37 C (Zeiss Axiolab Microscope HT-2000 Heating System) for assessment of ejaculate volume, sperm
concentration and motility characteristics.
A comprehensive CASA analysis was conducted using a SpermVision system. (Model 35, Minitube of America, Inc. WI). Several

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Table 1
Motion Characteristics of Sperm Cell Tracks.

Characteristic Attribute Description

VCL Linear Velocity Velocity over the total distance moved (in microns/sec), i.e., including all deviations of sperm head movement.
VAP Average Path Velocity Velocity over a calculated, smoothed path (in microns/sec), i.e., a shorter distance than that used for calculating VCL.
VSL Straight-Line Velocity Velocity calculated (in microns/sec) using the straight-line distance between the beginning and end of the sperm
track.
LIN Linearity Ratio of VSL/VCL describing path curvature (in% linearity)
STR Straightness Ratio between VSL/VAP (in% straightness)

studies have veried how CASA analyses of semen samples from a broad range of species, enables researchers to acquire objective
and precise information regarding the concentration of motile sperm cells within a sample, as well as distinguishing characteristics of
their motion patterns. The requisite motion-characteristic data compiled and tabulated in this study to investigate sperm viability
criteria included sperm concentration (x 109 per mL); total sperm (x 109); and proportion of motile spermatozoa within each sample
(Amann et al., 1980; Ranganathan et al., 2000).
CASA-derived motion characteristic data from individual sperm measurements provided signicant mean motility information
regarding signicance of progressively motile versus non-progressively motile sperm (Anzar et al., 1991; Budworth et al., 1988).
Additional criteria evaluated to determine sperm quality was assessed through measurement of three established sperm cell velocity
descriptors and their calculated ratios as detailed in Table 1. These specic motion parameters were obtained by analyzing a
minimum of 100 sperm tracks per sample.
The percentages of progressive motility and kinematic parameters (VCL, VAP, VSL, LIN, and STR) in this study were all quantied
using CASA-automated calculations. This provided quantitative data sets to compare the movement pattern characteristics of sperm
cells from each animals collected ejaculate.
Multiple elds containing up to 200 spermatozoa/eld were analyzed for each collected ejaculate utilizing CASA digital hard-
ware/software technology with integral digital microscopic video and photography. The SpermVision system initially assessed
sperm cell concentration, then
characterized cell motility through analysis of sperm movement sequences and velocity. The comprehensive data provided by
CASA motility analyses helped determine sperm viability through measurement of curvilinear velocity (VCL); the average path
velocity of the sperm head along its actual trajectory (VAP); straight-line velocity (VSL); the average path velocity of the sperm head
along a straight line from its rst to its last position; average path velocity (VAP); and the average velocity of the sperm head along its
average trajectory. Ratios of select parameters were then calculated to ascertain percentage of linearity (LIN), the ratio between VSL
and VCL; and the percentage of straightness (STR), the ratio between VSL and VAP.

2.7. Statistical analyses

Statistical analysis to validate quality of data assessments was performed using the SPSS analytics platform (IBM, New York).
Results data recorded for anesthetic and physiologic parameters, as well as semen characteristic criteria were calculated using the t-
distribution method expressed as mean SEM with a 95% condence interval (CI). A Pearson Correlation analysis was conducted in
Excel using the Analysis Toolpak add-in (Microsoft Corp., Washington) to determine the extent of any linear relationships between
variable data sets.

3. Results

Each animal was successfully anesthetized following BAM administration within its holding pen, then transported into the semen
laboratory in preparation for the electroejaculation procedure. The mean elapsed time from injection until time of recumbency was
10.2 0.92 min. All deer were observed to remain extremely calm and relaxed during the induction stage with no adverse events
reported. The butorphanol-azaperone-medetomidine combination provided a plane of anesthesia allowing for painless and ecient
semen collection.
Mean rectal body temperatures for animals in this study were 100.9 0.14F. This was well within the normal 95 F to 105 F
rectal temperature range of white-tailed deer (Nielsen, 1999).
During BAM anesthesia, median heart rate was 39.6 0.95 beats per minute. This compared favorably to heart rate ranges
reported in previous studies using BAM (Miller et al., 2009) as well as other immobilization drug combinations (Miller et al., 2003;
Storms et al., 2005).
Normal respiratory rates for deer at rest range from 16 to 20 breaths per minute (Nielsen, 1999).
Mean respiration rates for deer in this study remained within normal levels at 20.2 1.55 breaths per minute, throughout the
BAM anesthesia. This rate also corresponded well with previously published white-tailed deer studies using BAM for immobilization
(Miller et al., 2009; Siegal-Willott et al., 2009). A summary of measured physiological data is presented in Table 2.
To validate that use of a consistent 2.0 mL BAM dose rate volume regardless of the broad bodyweight range of test bucks did not
inuence measured results, Pearson correlation coecients were calculated for all recorded physiological parameters and semen
characteristics. To determine if changes in the BAM dose rate/animal weight (in mg/kg) expressed a linear relationship with the

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Table 2
Mean SEM temperature, heart rate and respiratory rate in adult white tailed deer.

Physiologic Parameters Unit Mean Values

Rectal temperature C 100.9 0.14

Heart rate beats minute-1 39.6 0.95
Respiratory frequency breaths minute-1 20.2 1.55

Data are presented as mean SEM.

examined variables of body temperature, heart and respiration rates along with semen volume, sperm concentration and motility,
statistical correlation coecients were analyzed. Our results indicated no correlation between the varied BAM anesthesia dose rates
and physiological/semen variable data sets.
Administration of atipamezole and naltrexone eectively reversed anesthesia produced by BAM with rapid, calm and persistent
recoveries. The mean recovery times (in min) from injection of antagonists until each animal was standing and walking normally was
3.4 2.32 min. Deer anesthetized with BAM (n = 10) showed no sign of sedation at ten minutes post-injection of antagonists
(Table 3). No re-sedation was observed in any of the test animals.
Based on the previously established Miller et al. (2009) cervid behavioral and quality of recovery rating system, scoring for all test
bucks undergoing BAM anesthesia (Table 4), correlated well with previously published white-tailed deer study results (Miller et al.,
2009; Storms et al., 2005).
Assessment of the studied sperm parameters collected from BAM immobilized animals in this study (volume of ejaculates, total
sperm concentration, motility and overall viability) indicated comparable semen quality as compared to other anesthetic protocols
used for electroejaculation (Fumagalli et al., 2012; Stewart et al., 2016).
Analyses of collected samples revealed a 1.1 0.37 mL mean seminal ejaculate volume, a sperm concentration (x109/mL) value
of 1.93 0.84 and a total sperm count mean of 1.94 109 0.71 (Table 5). As exemplied in the section below, these results are
well correlated with semen collection data reported in previously published cervid studies (Pintus and Ros-Santaella, 2014; Villagrn
and Ungerfeld, 2013).
Sperm cell motility from all collected samples was shown to be consistently elevated with an average value of 74.3% 0.14.
Sperm swimming velocity patterns recorded following CASA analysis revealed median values of VCL: 102.00 7.28 /sec, VAP:
58.70 3.34 /sec, and VSL: 47.20 2.90 /sec. The remaining kinematic parameters calculated from mean VCL, VAP and VSL
ratios determining percentage straightness and linearity were STR 80.4 0.86% and LIN 47.1 0.39%. (Table 6)

4. Discussion

This study demonstrates that ejaculates obtained from a single group of test bucks exhibited similar semen quality characteristics
as compared to other anesthesia protocols used for cervid immobilization. Overall, few dierences were observed as to induction of
anesthesia level for successful electroejaculation as compared to several published cervid studies (Garde et al., 2006; Martinez-Pastor
et al., 2009; Stewart et al., 2016).
These results indicate that sperm concentration, progressive sperm motility and other indicators of healthful sperm were similar
to values seen in earlier studies. (Malo, 2004; Ake-Lopez et al., 2010). In addition key kinematic motion pattern data recorded
correlated well with traits deemed important for sperm viability as established by Mortimer (Mortimer, 2000, 1997).
For comparison, mean sperm concentration in this study was 1.93 0.84 (x109/mL). Concentrations from previous semen
collection studies in cervids range from 1.321.75 (x109/mL) (Malo, 2004; Santiago-Moreno et al., 2011). Previous studies of other
anesthesia protocols for cervid electrojaculation including telazol/xylazine, detomidine/ketamine and ketamine/xylazine combi-
nations reported comparable levels of progressive sperm motility as this studys 74.3% ( 0.14). Results from other published trials
assessing percentages of progressive sperm ranged from 63.180.0% (Martinez-Pastor et al., 2009; Santiago-Moreno et al., 2011;
Stewart et al., 2016). Studies in various species including horses, dogs and rodents have described how sperm motility parameters are
eective indicators of viable sperm function and a primary determinant of successful fertilization. In one study (Scott, 2000) sperm
motility was found to be required for eective female oviduct colonization during the critical sperm transport phase.
Prior publications in deer and other mammals (TH Clutton-Brock, 1988; Wildt et al., 1987) have reported that three important
sperm velocity parameters (VCL, VAP and VSL) exhibit the most signicant associations with fertility rates. Beginning as early as

Table 3
Mean induction to recovery data for 10 adult male captive white-tailed deer (Odocoileus virginianus) with BAM.

Time to recumbency (min) Time duration of semen collection (min) Total down time (min) Time to recovery (min)

Mean 10.2 54.4 57.8 3.4

Range 6.015.0 40.0106.0 34.0108.0 1.08.0
SD 2.93 20.53 20.56 2.32
95% CI 8.1312.24 39.9368.87 43.3172.28 1.765.04
SEM 0.92 6.49 6.5 0.73

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Table 4
Ecacy and quality of recovery* following BAM immobilization, recorded at time 0, 3 and 5 min following reversal injections.

Time to recovery (min) Quality of recovery at 0 min. Quality of recovery at 3 min. Quality of recovery at 5 min.

Mean 3.4 5.0 0.8 0.6

SEM 0.73 0 0.51 0.38

*Quality of recovery recorded at time 0, 3 and 5 min following reversal injection, based on the following rating score criteria: 0 = no sign of sedation; 1 = standing
with minimal sedation/eyelids drooping; 2 = standing with moderate ataxia; 3 = unable to stand but head up; 4 = lateral unable to lift head; 5 = lateral re-
cumbency/no reversal.

Table 5
Summary of mean semen characteristics values for all test bucks.

Seminal volume (ml) 1.1 0.37

9
Sperm concentration (x10 /ml) 1.93 0.84
Total sperm count/ejaculate (x 109) 1.94 0.71
Progressive motility (%) 74.3 0.14

Data are presented as the mean SEM.

Table 6
Mean motility and velocity values calculated from all collected semen ejaculated of BAM-im-
mobilized test bucks

Primary Kinematic Sperm Motion Parameters BAM

(a) PMOT 74.3 0.14

(b) VCL (microns/sec) 102.00 7.28
(c) VAP (in microns/sec) 58.70 3.34
(d) VSL (in microns/sec) 47.20 2.90
(e) STR (% straightness) 80.4 0.86%
(f) LIN (% linearity) 47.1 0.39%

Comparative traits included progressive motility rate (a), linear velocity(b), average path ve-
locity (c), straight line velocity (d), straightness(e), and linearity (f).
Data are expressed as mean SEM.

1985, Blazek et al. determined how analysis of these parameters by CASA calculation of sperm motion tracks (as illustrated in Fig. 1),
directly correlated to dierences in fertility rates (Blazak et al., 1985). A 2004 study in red deer (Cervus elaphus) was designed to
identify specic semen traits indicating signicant associations with male fertility and conception rates in cervids (Malo, 2004). Their
analyses revealed a conclusive association between quality of semen production and the velocity parameters of spermatozoa
swimming at high speed (Malo, 2004).
Multiple studies have also established certain CASA-calculated benchmarks shown to correlate with fertilization success. Sperm
velocity has been found to be critical during the process of fertilization in a large number of mammalian species (Ros-Santaella et al.,
2014). Quantication of standard thresholds indicating attributes of fertile spermatozoa in deer, as well as horses and dogs have been
shown to exhibit: > 70% progressive motility; an average path velocity of > 50 m/second; and > 75% straightness level (Dascanio
and McCue, 2014; Loomis and Graham, 2008; Malo, 2004).
VAP, VCL, and VSL data from this current study are closely correlated to the velocity parameters reported in various cervid
fertility studies (Martnez, 2008; Ramn et al., 2013).
A recent red deer fertility study (Ramn et al., 2013) reported that males with high fertility rates had ejaculates containing high
percentages of spermatozoa exhibiting rapid and linear movement. Mean assessments in this study of average path velocity (VAP),

Fig. 1. Criteria and formula models for analysis of individual sperm head motion tracks.

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percent straightness (STR) and linearity (LIN) of sperm cell motion patterns all revealed values within well-established ranges of
successful fertilization capacity (Jimnez-Rabadn et al., 2012; Loomis and Graham, 2008; Ramn et al., 2013).
In conclusion, the results of this study suggest butorphanol-azaperone-medetomidine (BAM) anesthesia provides eective an-
esthesia for successful semen collection in white-tailed bucks. The results of assessed sperm concentrations of collected ejaculates,
sperm motility and kinematic motion parameters demonstrated that the collected samples exhibited requisite levels of the sperm cell
viability established for successful fertilization (Loomis and Graham, 2008; Malo, 2004). Further studies should be conducted using
semen collected following BAM immobilization to determine eects on semen freezing and thawing characteristics, as well as as-
sessment of resulting conception and birth rates.

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