Computer assisted sperm analysis of motility patterns of postthawed epididymal spermatozoa of springbok (Antidorcas marsupialis), impala (Aepyceros melampus), and blesbok (Damaliscus dorcus phillipsi) incubated under conditions supporting domestic cattle in vitro fertilization

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Theriogenology 78 (2012) 402– 414 www.theriojournal.com

Computer assisted sperm analysis of motility patterns of postthawed epididymal spermatozoa of springbok (Antidorcas marsupialis), impala (Aepyceros melampus), and blesbok (Damaliscus dorcus phillipsi) incubated under conditions supporting domestic cattle in vitro fertilization F.P. Chatizaa,*, P. Bartelsb, T.L. Nedambalec, G.M. Wagenaara a b

University of Johannesburg, Department of Zoology, Auckland, Park Johannesburg, South Africa 2006 Wildlife Biological Resources of National Zoological Gardens, Pelindaba, Pretoria, South Africa 0001 c Agricultural Research Council, Animal Production Institute, Irene, South Africa 0062 Received 29 July 2011; received in revised form 16 February 2012; accepted 19 February 2012

Abstract The need for information on the reproductive physiology of different wildlife species is important for ex situ conservation using such methods as in vitro fertilization (IVF). Information on species reproductive physiology and evaluation of sperm quality using accurate, objective, repeatable methods, such as computer-assisted sperm analysis (CASA) for ex situ conservation has become a priority. The aim of this study was to evaluate motility patterns of antelope epididymal spermatozoa incubated for 4 h under conditions that support bovine IVF using CASA. Cauda epididymal spermatozoa were collected postmortem from testicles of springbok (N ⫽ 38), impala (N ⫽ 26), and blesbok (N ⫽ 42), and cryopreserved in biladyl containing 7% glycerol. Spermatozoa were thawed and incubated in Capacitation media and modified Tyrode lactate (m-TL) IVF media using a protocol developed for domestic cattle IVF. The study evaluates 14 motility characteristics of the antelope epididymal sperm at six time points using CASA. Species differences in CASA parameters evaluated under similar conditions were observed. Several differences in individual motility parameters at the time points were reported for each species. Epididymal sperm of the different antelope species responded differently to capacitation agents exhibiting variations in hyperactivity. Motility parameters that describe the vigor of sperm decreased over time. Spermatozoa from the different antelope species have different physiological and optimal capacitation and in vitro culture requirements. The interspecies comparison of kinematic parameters of spermatozoa between the antelopes over several end points contributes to comparative sperm physiology which forms an important step in the development of species specific assisted reproductive techniques (ARTs) for ex situ conservation of these species. © 2012 Elsevier Inc. All rights reserved. Keywords: Antelope; Protocols; Ex situ conservation

1. Introduction

* Corresponding author. Tel.: ⫹263 772 269 230; fax: ⫹27 12 305 5840 or ⫹27 11 559 2286. E-mail address: [email protected] (F.P. Chatiza). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2012.02.020

The loss of biodiversity on wildlife populations is a cause for concern to conservationists worldwide. Anthropogenic practices that cause loss of wildlife diversity, genetic variability, and population decline in wild

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animals include population fragmentation through land transformation, habitat fragmentation, and alteration resulting in habitat loss, excessive consumptive utilization and predation [1–5]. To limit the loss of biodiversity and the negative effect of small and fragmented populations, there is an urgent need for the development and use of all means available which may include the use of ex situ procedures to conserve species outside their natural habitat. This includes the use of captive breeding programs and assisted reproductive techniques (ARTs) to preserve and use gametes and genomic material from different species. This initiative has become progressively important, considering the possibilities that this approach offers. Conservation efforts that promote genetic variability in both in situ and ex situ populations are therefore highly recommended [6]. A large component of assisted reproductive technique conservation is dependent on knowledge of the reproductive biology and physiology of different species and use of reproduction protocols for the transfer of genetics [5,7–9]. Therefore, this research will evaluate sperm quality and function based on the evaluation of sperm motility parameters using computer assisted sperm analyzer by means of the sperm class analyzer (CASASCA). Motility is the most commonly used parameter in the evaluation of frozen sperm [10] and predicting the fertilizing capacity of animals [11,12]. Sperm motility reflects several essential aspects of sperm metabolism, and is a readily assayed barometer of relative cell health [13]. In equine, longevity of motility evaluated using light microscopy has been significantly correlated with the foaling rate [14]. Some authors suggest that conventional sperm analysis is sufficient to evaluate the fertility potential of semen [12]. However conventional evaluation which is based on subjective visual measurements may underestimate good motility in some species [13]. Studies have also demonstrated a large variation between and within technician in evaluating motility. This has resulted in inaccurate and imprecise measurements of sperm motility [15] and a considerable variation in sperm quality measurements amongst laboratories, processing methods, and microscopic analysis of sperm samples. This makes data incomparable, and interpretation of results complex [16] particularly when working with wildlife samples. These limitations imply that conventional motility should be evaluated together with other parameters to obtain a better estimate of sperm fertilizing potential [17–19]. Computer assisted sperm analysis (CASA) therefore provides a more re-

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liable accurate and objective method of evaluating motility while facilitating a rapid analysis of motility patterns. CASA systems, such as the SCA can greatly assist to standardize methodologies. The SCA produces comprehensive kinematic data for sperm motility which could be used to define sperm which vary in their motility track or trajectory [20,21] providing comprehensive information on sperm quality in terms of motility than just overall motility. The SCA can therefore be used to monitor changes in motility patterns over time thus giving insight into the physiological characteristics of sperm in different in vitro environments. In an effort to contribute to ex situ conservation of selected South African antelope and understanding the physiology of their sperm, the aim of this study is therefore to evaluate changes in motility patterns of postthawed epididymal sperm subjected to a protocol that supports domestic cattle in vitro fertilization (IVF) by means of CASA-SCA. 2. Materials and methods Chemicals used in this study were obtained from Sigma, South Africa unless otherwise stated. 2.1. Testes collection and sperm extraction Testes were collected postmortem from sexually mature, free ranging springbok (N ⫽ 38), impala (N ⫽ 26), and blesbok (N ⫽ 42) during organized winter culls in the period May to July in South Africa. Animals were obtained in their breeding season from different game farms and reserves throughout South Africa. Culls or hunts were conducted or supervised by professional hunting teams or staff. None of the animals had been born captive or hand-raised. All animals were killed for management purposes on the game farms or reserves. The paired testes from each animal were removed and transported for sperm processing in closed plastic bags to an onsite mobile laboratory within 3 h of death in a cooled styrofoam container. Testes were placed on cardboard under which were ice bricks avoiding contact with ice bricks but maintaining a temperature of between 4 °C and 5 °C. The epididymis and proximal vas differentia were detached from both testes. The cauda epididymis and vas deferens from each animal were detached from the caput and corpus epididymis. A blunt 26-gauge needle was inserted into the lumen of the vas deferens for sperm harvesting. The needle was attached to a syringe containing 2 mL of Biladyl A extender (Minitüb, Tiefenbach, Germany) consisting of 2.42% Tris, 1.38% citric

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acid, 1% fructose, and supplemented with 20% egg yolk (pH 6.74). Spermatozoa were flushed out of the cauda epididymis by applying pressure through the syringe. Sperm extraction was carried out at ambient temperature (10 °C to 4 °C). 2.2. Sperm processing and cryopreservation Spermatozoa from both testes of an individual animal were pooled to make a single sample unit if the sperm motility (determined subjectively using 100⫻ light microscopy) was similar from both testes. The total concentration of each pooled sample was determined using a hemocytometer. When the total concentration was determined, samples were then diluted with Biladyl A (Minitüb) at ambient temperature to a concentration of 400 ⫻ 106 sperm/mL. The extended spermatozoa were placed in 50 mL falcon tubes which were then placed in a water bath at room temperature (20 °C to 25 °C) and slowly cooled to 4 °C in a cold room for 6 h. An equal volume of Biladyl B (Minitüb) at 4 °C containing 14% glycerol was then added slowly drop by drop in a single step to each sample resulting in a 1:1 dilution of the original sample and a final glycerol concentration of 7%. Samples with glycerol were mixed and equilibrated for an additional 20 min at 4 °C before loading into straws. Sperm samples were then loaded into labeled 0.25-mL plastic straws, sealed, and placed 4 cm above liquid nitrogen in liquid nitrogen vapors (⫺75°C and ⫺125°C) for 20 min after which straws were immediately plunged into liquid nitrogen (LN2, ⫺196 °C) and held for at least 5 min in liquid nitrogen before transferring into liquid nitrogen tanks for storage for at least 6 mo before thawing. Cryopreservation of spermatozoa was carried out under field conditions in a mobile laboratory. Sperm extraction and processing were carried out at room temperature unless otherwise stated. 2.3. Thawing and processing of postthawed antelope epididymal sperm under conditions that support domestic cattle IVF Straws containing cryopreserved sperm were removed from liquid nitrogen tanks, briefly thawed in air for 10 sec then plunged into a water bath at 37 °C for 20 sec, wiped with tissue sprayed with 70% ethanol solution and finally dried with tissue. The thawed spermatozoa from each straw were then released into a round-bottom centrifuge tube (“swim-up tube”). A small drop of the thawed sperm was placed onto a

microscope slide to check motility, which was determined subjectively using 100⫻ light microscopy. Once motility was confirmed, 85 ␮L of sperm was layered under 1 mL of Capacitation medium (Modified Tyrode medium) [22,23] without CaCl2-2H2O and supplemented with 0.9 mM sodium pyruvate, 6 mg/mL “fatty acid free” BSA, 6.9 mM glucose, 4 mM sodium lactate, and 50 mg/mL penicillin/streptomycin per tube [23]. Three tubes were prepared for “swim-up” and incubated for 30 min at 38 °C and 5% CO2 in a humidified atmosphere Sanyo incubator. After 30 min the top layer containing live/motile sperm was transferred into a clean conical tube and centrifuged for 10 min at 300 ⫻ g. After 10 min the supernatant was discarded and the pellet of sperm was resuspended in 20 ␮L of Capacitation medium (After Cent). This fraction was then diluted in 100 ␮L of domestic cattle modified TyrodeLactate IVF medium (m-TL) [22,23] supplemented with 6 mg/mL “fatty acid free” BSA, 0.9 mM sodium pyruvate, 0.25 mg/mL heparin, 0.125 mg/mL hypotaurine, and 0.125mg/mL epinephrine, and incubated in closed eppendorf tubes with perforated lids at 38 °C and 5% CO2 in humidified atmosphere incubator for a total of 4 h. 2.4. Evaluation of motility patterns of postthawed epididymal sperm incubated under conditions that support domestic cattle IVF using CASA Motility patterns of the postthawed antelope cauda epididymal sperm were subjected to different processing stages: (1) “swim-up” in Capacitation medium, (2) centrifugation, and (3) culture in m-TL-IVF medium of a domestic cattle IVF protocol as previously stated. Motility patterns of the antelope cauda epididymal spermatozoa were determined using a deer program on at least 200 cells per sample in a minimum of four fields by CASA using an SCA (V.4.001). Five ␮L of sperm suspension was placed on a prewarmed microscope slide, overlaid with a 22 mm2 prewarmed coverslip and the slide maintained at 37 °C during analysis by a heated slide warmer. Sperm motility was video taped using a Basler camera attached to a Nikon Eclipse microscope equipped with a 10⫻ negative phase-contrast objective and a 10⫻ projector ocular. The video tape was replayed for analysis at a subsequent time and the playback feature was used to identify and delete aberrant tracks occurring when trajectories cross over each other or spermatozoa collide. Instrument setting for the CASA analysis were as follows: number of images: 50; images per sec: 50; optics phase-contrast, scale 10x; particle area: 5 to 80 ␮m; ⬍20 ␮: slow; ⬍60

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␮: medium; ⬍120 ␮: rapid; progressivity ⬎80% straightness (STR); circular ⬍50%: linearity (LIN); connectivity: 12; restrictions warning to 3 fields; 500 sperm; 20% concentration; image in report: 15. The following motility characteristics were determined; progressive motility (PM), nonprogressive motility (NPM), static (STC), rapid (RAP), medium (MED), slow (SLOW), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), LIN, wobbling (WOB), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), and STR in these fractions (1) immediately upon layering sperm with 1 mL of Capacitation medium (“swim-up” [Sw] 0 min), (2) in the top fraction after “swim-up” for 30 min (Sw 30 min), (3) in resuspended pellet after centrifugation for 10 min (After Cent), (4) at intervals (0 h, 2 h, 4 h) during incubation in m-TL IVF medium denoted as m-TL 0 h, m-TL 2 h, m-TL 4 h. 2.5. Statistical analysis SPSS version 18.0 (Statcon, South Africa) was used for statistical analyses. One-way analysis of variance (ANOVA) (generalized linear model; GLM) was used for the comparison of the sperm motility between species. Values were expressed as mean ⫾ standard error of mean (SEM). Significance level was set at P ⬍ 0.05. Comparison of interspecies motility patterns evaluated using CASA-SCA was made using nonparametric test. Shapiro-Wilks test for normality showed that data were not normally distributed for all species and there were no equal variances. Kruskal-Wallis test was used to determine differences between treatments/variables, significance level was set at P ⬍ 0.05. The MannWhitney test was used to determine differences between species for respective treatments/variables. Significance level was set at P ⬍ 0.0167. 3. Results 3.1. Total motility Total motility differed significantly between species (P ⬍ 0.05) immediately after thawing with blesbok exhibiting the highest total motility (86%) and impala the lowest total motility (58%) (Fig. 1). Upon incubation (0 h) in m-TL IVF media total motility was 79 ⫾ 3.01% for springbok, 72 ⫾ 2.12% for impala, and 63 ⫾ 1.98% for blesbok. On average, total motility at the time of insemination was ⬎71% across species however percentage motility decreased over time. Total motility increased (P ⬍ 0.05) from Sw 0 h to m-TL 0 h

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Fig. 1. Total motility of postthawed cauda epididymal sperm springbok, impala, and blesbok immediately after thawing. Species with common letters do not significantly differ from each other.

for springbok and impala however motility declined for blesbok. 3.2. Progressive motility (PM), nonprogressive motility (NPM), static (STC) Species behaved differently across the treatments immediately upon layering epididymal sperm under “swim-up”/capacitation media (Sw 0 min); after “swim-up” for 30 min (Sw 30 min); after centrifugation (After Cent); after incubation in m-TL IVF media at 0 h, 2 h, 4 h (m-TL 0 h; m-TL 2 h; m-TL 4 h) and over time in m-TL IVF media with regards to PM, NPM, and STC (Table 1). PM varied between species across treatments with a general decline in PM over time. Blesbok had the highest PM (P ⬍ 0.0167) of the three species. PM increased after Sw 30 min for springbok and impala with springbok exhibiting the highest increase in PM after Sw 30 min and consequently had the highest PM (P ⬍ 0.0167) after swim-up. However blesbok exhibited a decline in PM from Sw 30 min to m-TL 4 h. PM decreased over time in m-TL IVF media for springbok and impala but the decrease was smaller for blesbok. Impala had the highest initial PM (P ⬍ 0.0167) in m-TL IVF media. Impala had the largest decline in PM from m-TL 0 h to m-TL 2 h. PM was lower than NPM and STC across treatments for all species. NPM differed (P ⬍ 0.0167) between species with a gradual decrease across treatments and over time in m-TL IVF media. Blesbok showed a significant increase (P ⬍ 0.05) in NPM of the three species after centrifugation. Percentage STC gradually increased across treatments for all species with impala maintaining the highest STC (P ⬍ 0.05) across treatments of the three species (Fig. 2).

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Table 1 Results of Mann-Whitney test indicating which species differ for the respective motility patterns. Motility pattern PM NPM STC LIN STR WOB

RAP

Sw 0

Sw 30

Sp vs. bl imp vs. bl Sp vs. bl Sp vs. bl imp vs. Sp vs. bl imp vs. Sp vs. bl imp vs. Sp vs. bl imp vs.

Sp vs. imp NS Sp vs. imp

bl Sp vs. bl bl NS bl Sp vs. bl bl

After cent Sp vs. bl imp vs. bl Sp vs. bl imp vs. bl Sp vs. imp imp vs. bl Sp vs. bl imp vs. bl Sp vs. bl imp vs. bl Sp vs. bl imp vs. bl

Sp vs. imp sp vs. bl

NS

MED

Sp vs. imp sp vs. bl imp vs. bl NS

Sp vs. bl

Sp vs. bl

SLOW

NS

Sp vs. bl

Sp vs. bl

VAP

Sp vs. bl imp vs. bl Imp vs. bl

NS

NS

NS

NS

VCL

VSL

Sp vs. bl imp vs. bl

imp vs. bl

Sp vs. imp imp vs. bl

ALH

NS

Sp vs. bl

BCF

Sp vs. bl imp vs. bl

Sp vs. imp imp vs. bl

Sp vs. bl imp vs. bl Sp vs. imp sp vs. bl imp vs. bl

m-TL 0 h

m-TL 2 h

m-TL 4 h

Sp vs. imp

Sp vs. imp

Sp vs. imp

Sp vs. imp imp vs. bl Sp vs. imp imp vs. bl Sp vs. imp imp vs. bl Sp vs. imp

NS

NS

NS NS

Sp vs. imp imp vs. bl NS

NS

NS

Sp vs. bl

Sp vs. imp sp vs. bl

Sp vs. imp sp vs. bl

Sp vs. imp sp vs. bl

NS

Sp vs. imp

NS

NS

Sp vs. imp vs. bl Sp vs. imp sp vs. bl

Sp vs. bl

Sp vs. imp sp vs. bl imp vs. bl Sp vs. bl imp vs. bl Sp vs. imp sp vs. bl Sp vs. bl sp vs. imp imp vs. bl Sp vs. imp sp vs. bl Sp vs. imp sp vs. bl imp vs. bl Sp vs. imp sp vs. bl imp vs. bl Imp vs. bl sp vs. bl Sp vs. imp imp vs. bl

Sp vs. bl

Sp vs. imp

Sp vs. imp

NS

Sp vs. imp sp vs. bl NS

NS

Significance level P ⬍ 0.0167. Significant difference between springbok and impala is indicated by sp vs. imp; significant difference between springbok and blesbok is indicated by sp vs. bl; and significant difference between impala and blesbok is indicated by imp vs. bl. NS represents no significant difference between species. After cent, Capacitation medium; ALH, amplitude of lateral head displacement; BCF, beat cross frequency; LIN, linearity; MED, medium; m-TL, modified Tyrode Lactate; NPM, nonprogressive motility; PM, progressive motility; RAP, rapid; SLOW, slow; STC, static; STR, straightness; Sw, incubated in capacitation media;VAP, average path velocity; VCL, curvilinear velocity; VSL, straight line velocity; WOB, wobble.

3.3. Linearity (LIN), straightness (STR), wobbling (WOB) Species behaved differently across treatments and over time in m-TL IVF media with regards to LIN, STR, and WOB (Table 1). LIN differed between species and across treatments. Springbok and impala exhibited a steady increase in LIN from Sw 0 min to m-TL 0 h, however blesbok exhibited a decline in LIN from Sw 0 min to IVF 0 h. LIN was highest (P ⬍ 0.0167) in the following treatments: Sw 0 min, IVF 0 h, m-TL 2 h, for blesbok, impala, and springbok, respectively. LIN appeared to be lower than STR and WOB

across treatments for all species. STR and WOB differed (P ⬍ 0.0167) between species and across treatments. Except for blesbok (After Cent), STR and STC were not significantly different (P ⬎ 0.05) across treatments for all species (Fig. 3). 3.4. Rapid (RAP), medium (MED), slow (SLOW) Species behaved differently across treatments and over time in m-TL IVF media with regards to RAP, MED, and SLOW (Table 1). Blesbok had the highest RAP (P ⬍ 0.0167) at Sw 0 min after which RAP declined (P ⬍ 0.0167) drastically after Sw 30 min in

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treatments and over time in m-TL IVF media for springbok and impala (Fig. 4). 3.5. Average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL) Species behaved differently across treatments and over time in m-TL IVF media with regards to VAP, VCL, and VSL (Table 1). Blesbok exhibited the highest VAP (P ⬍ 0.0167) at Sw 0 min. For springbok and impala, VAP steadily increased after Sw 30 min. VAP generally declined over time in m-TL IVF media for all species. VCL

Fig. 2. Progressive motility (PM), nonprogressive motility (NPM), and static (STC) of post-thawed springbok, impala and blesbok cauda epididymal sperm of incubated in capacitation (Sw) media and modified Tyrode Lactate (m-TL) media following a protocol supporting domestic cattle IVF. Aft Cent, Capacitation medium.

comparison with springbok and impala. However RAP increased after Sw 30 min for springbok and impala before declining After Cent. After cent RAP increased for springbok. RAP declined over time in IVF media for all species. Springbok and impala exhibited the highest RAP (P ⬍ 0.0167) in m-TL IVF media at 0 h with no difference in RAP (P ⬎ 0.0167) between them. Species behaved differently in terms of MED across treatment and over time with the highest MED exhibited after centrifugation. Blesbok maintained the highest MED over time. Generally MED declined over 4 h in m-TL IVF media for all species. Blesbok maintained the highest SLOW across treatments and over time in m-TL IVF media but the difference between species was not statistically significant in some categories. After Sw 30 min, SLOW remained fairly constant across

Fig. 3. Linearity (LIN), straightness (STR), and wobble (WOB) of postthawed springbok, impala, and blesbok cauda epididymal sperm of incubated in capacitation (Sw) media and modified Tyrode Lactate (m-TL) media following a protocol supporting domestic cattle IVF. Aft Cent, Capacitation medium.

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3.6. Amplitude of lateral head displacement (ALH) Species behaved differently across treatments and over time with regards to ALH (Table 1) with a general decline in ALH across treatments and over time in m-TL IVF media for all species. Springbok and impala exhibited the highest ALH (P ⬍ 0.0167) at m-TL 0 h. ALH declined gradually from Sw 0 min to m-TL 4 h for blesbok. Blesbok had the lowest ALH of the antelope species across treatments except at m-TL 4 h where impala exhibited the lowest ALH. The rate of decline in ALH was steeper in m-TL IVF media for all species compared with the preceding treatments (Fig. 6).

Fig. 4. Rapid (RAP), medium (MED), and slow (SLOW), of postthawed springbok, impala, and blesbok cauda epididymal sperm of incubated in capacitation (Sw) media and modified Tyrode Lactate (m-TL) media following a protocol supporting domestic cattle IVF. Aft Cent, Capacitation medium.

was higher than VAP and VSL across treatments for all antelope species. Species behaved differently in terms of VCL with springbok and impala showing an increase in VCL from Sw 0 min to m-TL 0 h and exhibiting the highest VCL at IVF 0 h. Impala exhibited the highest (P ⬍ 0.0167) VCL at m-TL 0 h. In m-TL IVF media, VCL declined over time for all species with differences occurring between species at each time point. VSL exhibited the lowest velocities across treatments and over time for all species. Species behaved differently over time in m-TL IVF media in terms of VSL however species exhibited a decline in VSL over time (Fig. 5).

Fig. 5. Average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL), of post-thawed springbok, impala, and blesbok cauda epididymal sperm of incubated in capacitation (Sw) media and modified Tyrode Lactate (m-TL) media following a protocol supporting domestic cattle IVF. Aft Cent, Capacitation medium.

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highest RAP values exhibited by springbok and impala (54%) and a minimum of 17% by blesbok; the highest VCL values of exhibited by springbok and impala (161 to 194 ␮m/sec, respectively) and least VCL (114 ␮m/ sec) exhibited by blesbok. Overall species behaved differently across treatments and over a period of 4 h (Table 1). 4. Discussion Fig. 6. Amplitude of lateral head displacement (ALH) of postthawed springbok, impala, and blesbok cauda epididymal sperm of incubated in capacitation (Sw) media and modified Tyrode Lactate (m-TL) media following a protocol supporting domestic cattle IVF. Aft Cent, Capacitation medium.

3.7. Beat cross frequency (BCF) Species behaved differently across treatments and over time with regards to BCF (Table 1) with a general decline in BCF across treatments and over time in m-TL IVF media for all species (Fig. 7). 3.8. Hyperactivity and capacitation Values defining hyperactivity and capacitation of antelope sperm are as follows: VCL; after “swim-up” in Capacitation media (105 to 143 ␮m/sec) with VCL increasing to between 114 and 195 ␮m/sec in m-TL IVF media. Highest values of BCF ranged from 12 to 16 Hz, and ALH ranged from 2.1 to 3.6 ␮m in m-TL IVF media across species with blesbok having the lowest BCF and ALH. Springbok maintained the highest BCF (11 to 16 Hz) over time in IVF media. Species variability was exhibited in VCL and RAP with the

Fig. 7. Beat cross frequency (BCF) of postthawed springbok, impala, and blesbok cauda epididymal sperm of incubated in capacitation (Sw) media and modified Tyrode Lactate (m-TL) media following a protocol supporting domestic cattle IVF. Aft Cent, Capacitation medium.

CASA presents an array of different kinematic parameters that are more informative in defining motility of sperm and overall sperm quality. CASA overcomes the limitations associated with using conventional and subjective evaluation of motility which is prone to human error and bias by increasing the objectivity, accuracy, and reproducibility of motility measurements and appreciation of the functional status of sperm [24,25]. CASA is also the only way to assess velocity and linearity. Of the motility parameters evaluated in this study VAP, VCL, VSL, ALH, BCF, and LIN are the most commonly evaluated CASA parameters in most studies [26] due to their significance as fertility indicators. In this study VAP, VCL, ALH, BCF, and RAP exhibited elevated values upon incubation in m-TL IVF media containing heparin, a capacitation agent, decreasing gradually over time while VSL, LIN, and PM had lower values in comparison. The parameters VAP, VCL, ALH, BCF, and RAP are associated with hyperactivated motility and capacitation and indicate specific aspects of sperm function, including capacitation and interaction with the zona pellucida [18]. Elevated VCL values are characteristic of sperm incubated under capacitating culture conditions and indicative of sperm undergoing vigorous hyperactivated pattern of activity characterized by high amplitude flagella bending. This study indicates that spermatozoa from the different antelope species responded to capacitation agents exhibiting hyperactivation. Hyperactivated motility of sperm has also been reported in other species, such as rat [26], hamster [27], human [28], and nonhuman primates [29] incubated under capacitating conditions. Hyperactivated motion is a type of vigorous nonlinear motion that mammalian sperm exhibit as they progress through the female oviduct [30,31]. During hyperactivation the pattern and vigor of sperm track undergo dramatic changes which are characterized by movements in a random path and result in a nonprogressive circular movement. These changes in motion are characterized by a change in flagella activity characterized

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by a whiplash flagella activity which is a natural event of capacitation [26]. Hyperactivation is characterized by the transition from a moderately active, relatively linear swimming pattern to a highly vigorous, nonprogressive random motion with high curvature flagella movements [32]. Other authors characterized hyperactivation by increases in lateral head displacement, flagella bend amplitude, and beat asymmetry [33,34]. Therefore, the tracks for hyperactivated sperm are expected to have increased ALH and VCL and decreased LIN and STR [26,35]. Values for ALH and VCL in this study concur with this assumption for all three antelope species. In vivo hyperactivation allows sperm to detach from the oviductal epithelium, is necessary for sperm to reach the site of fertilization in vivo [36], and provides increased thrust for penetration of the cumulus [37,38] and zona pellucida [39]. It is believed that hyperactivated motile sperm is signifies the final stage or completion of capacitation [40]. Sperm of many mammalian species are not ready to fertilize oocytes immediately after ejaculation or upon recovery from the epididymis. Therefore, sperm need to undergo capacitation either in vivo in the female reproductive tract or in vitro in conditioned culture medium to obtain the capacity to interact with oocytes [30,41]. During capacitation both in vivo and in vitro biochemical and molecular changes in the sperm plasma membrane occur, including changes in the pattern of sperm motility indicating hyperactivation [30,42]. According to Sukcharoen [43] the most important movement for IVF success is the incidence of hyperactivation after 3 h. In support of this McPartlin [35] demonstrated that a low level of hyperactivation is associated with poor fertilization rate. While hyperactivation and capacitation are influenced by media composition on the one hand, hyperactivity in sperm by contrast can also occur under noncapacitating conditions in some species, such as the golden hamster sperm incubated in media devoid of capacitating agents [44]. This is supported by several authors who reported that capacitation and hyperactivation are separable and independent events [29,35]. Species differences in VCL, BCF, and ALH in IVF media indicate that the response to capacitation agents varied between species. Potential species differences in sperm plasma membrane composition may account for the differences in the degree of capacitation as exhibited by the differences in ALH, VSL, LIN, and STR between the antelope species. The plasma membrane of sperm is unique within species with regard to phospholipid composition (approximately 70% phospholipids, 25% neutral lipids, and 5% glycolipids) as reported by

Flesh and Gadella [45] and Meyers [46]. Spermatozoa from the different species vary in their lipid composition, which affects the capacitation rates [47]. Some studies indicate that the cholesterol/phospholipid ratio of the plasma membrane is a major determinant in plasma membrane fluidity [48]. During capacitation, cholesterol efflux is necessary to increase membrane fluidity and to stimulate tyrosine phosphorylation. Increases in intracellular calcium and cholesterol efflux have been observed with capacitation and with cryopreservation [49 –52]. During capacitation, cholesterol efflux from the sperm membrane increases fluidity and permeability of the membranes leading to membrane fusion and acrosome reaction [53,54]. Sperm from species that posses very high cholesterol/phospholipids ratios, such as human and rabbit sperm, experience a lesser degree of fluidity, capacitation, and membrane damage [55–57]. A major cause of loss of acrosome integrity after freezing is cold shock, which results in swelling and blebbing of the acrosomal membrane and disruption and/or increased permeability of the plasma membrane [58]. The mechanism for loss of acrosome integrity results from the loss of cholesterol from the plasma membrane which is one of the most detrimental stages of capacitation causing an increase in permeability to Ca2⫹, HCO3, and K⫹ [51,59,60]. This cholesterol efflux and influx of high intracellular concentrations of these ions causes the plasma membrane to become unstable enhancing its ability to fuse with the outer acrosome resulting in acrosome reaction and membrane damage [48]. Both an increase in intracellular calcium and cholesterol efflux are observed with sperm capacitation and with cryopreservation [50,51]. Therefore, cryopreserved sperm have relatively high acrosome reaction and undergo changes that are similar to those occurring during capacitation. These capacitation-like cellular modifications reduce the reproductive lifespan and longevity of cryopreserved spermatozoa in the female reproductive tract particularly in species that have long estrus and require more careful timing of ovulation and insemination [61]. Variation among species susceptibility to cold shock and cryopreservation has been reported by several authors who have correlated this with variations in membrane lipid composition between sperm of different species. Cold shock resistance is generally higher for species with sperm membranes characterized by a greater sterol/cholesterol-to-phospholipid ratio [53,62] suggesting that different species may have different requirements for low-temperature storage.

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Formulating sperm wash and fertilization medium in vitro necessitates, including chemical components that sustain sperm motility, such as hypotaurine that promote capacitation, the union of gametes and the initiation of embryo development [44]. These include caffeine, heparin [23], glucose [63], and bicarbonate [64]. In vitro, capacitation of sperm is promoted by different chemical agents, such as bicarbonate, serum, BSA, heparin, caffeine, or glucose incorporated in IVF media. These chemical agents also have been used to support capacitation in bull [59], goat [65], mouse [66], and stallion [67] IVF systems. Different authors agree that motility patterns that characterize hyperactivation and capacitation appear to vary among species. For example, a study by Cancel et al. [26] on rat epididymal sperm incubated for 4 h under conditions that support IVF and capacitation demonstrated that VCL, BCF, and ALH increased while VSL, LIN, and STR decreased over time. On the contrary, antelope epididymal sperm showed a decrease in BCF over time across antelope species, LIN was variable over time decreasing after 4 h across species except for impala which showed a decrease over time in m-TL IVF media. Hyperactivated rat sperm exhibited the following values for the respective motility patterns increased values of VAP: 219 ␮m/sec; VCL: 653 ␮m/ sec; ALH: 35 ␮m; BCF: 30 Hz; and decreased values of VSL: 58 ␮m/sec; STR: 27%; and LIN: 9%. Hyperactivated stallion sperm is defined by VSL ⬍ 46.5 ␮m/ sec; STR ⬍ 46.6%; and LIN ⬍ 20.2%. In humans VAP ⬎ 25 ␮m/sec and STR ⬎80 is categorized as rapid progressive motile pattern characterized with high fertilization potential [68]. Species differences in motion characteristics for similar events highlighted above and the species differences in CASA values for sperm incubated under similar conditions implies differences in in vitro requirements and sperm physiology and may imply possible variations in fertility rate between sperm of different species. Values of PM, VCL, and RAP were variable between species with PM, VCL, and BCF increasing only for impala after centrifugation. Motility patterns also varied over time in m-TL IVF media between the different antelope species. Sperm motility parameters that indicated straightness, i.e., VSL, LIN, and STR, as well as VAP and ALH exhibited variability between species and over time. VAP values ranged from 78 to 99 ␮m/sec with springbok having the highest VAP and blesbok the lowest VAP upon incubation in m-TL IVF media. STR ranged from 67% to 72% between the antelope species upon incubation in m-TL IVF media.

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Blesbok appeared to have the lowest values in terms of VCL, RAP, ALH, and beat cross frequency of the three species. Variation in CASA parameters between species indicates that the rate of metabolic activity of sperm of the different antelope species in m-TL IVF media varies. The rate of metabolic activity is genetically predetermined, which confirms that spermatozoa of the different species may vary in this respect. Due to limited information on species specific protocols resulting from limited access to samples sperm cryopreservation and IVF in antelope have been carried out using protocols developed for domestic species, primarily cattle [69,70], with little or no modification. These methods have been fairly successful however when a cryopreservation or IVF protocol has been optimized for sperm of one species, it may not be ideal for sperm of other species as observed between bovine and caprine semen [58,60,71–73]. Therefore, this study adds to knowledge on comparative sperm physiology generated from kinematic studies that can lead to the optimization of protocols for individual species resulting in higher success cryopreservation and IVF rates for each antelope species. Variability among species in CASA parameters recorded under similar conditions implies that different motility parameters obtained by CASA may be correlated with fertility [42,74,75] in different species. This provides justification for further study of the correlation between CASA parameters and fertility in antelope. Total motility, VCL, and VSL have been correlated with fertility in bulls [42,74,75] VAP and VCL and ALH have been correlated with fertility the in Catalonian donkey [76] VCL and VAP [77], VCL, VSL ALH and hyperactivity [75] have been correlated with IVF results in humans with variability in CASA values between different studies. ALH has also been strongly correlated with sperm-oocyte fusion and polypronucleate zygote formation in humans [45,74]. On the contrary, VCL, LIN, ALH, total motility, and RAP had no significant correlation with fertility in a study on mares [78]. Variability among species in CASA parameters recorded under similar conditions also implies that spermatozoa of different species may have different physiological requirements and thus different optimal conditions for fertilization. A decrease in most CASA parameters over time was observed across antelope species. Generally PM, NPM, RAP, and MED, decreased over time particularly in IVF media. CASA parameters that describe the vigor of sperm VAP, VCL, ALH, and BCF also decreased over time in IVF media indicating a decrease in metabolic

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rate, adenosine triphosphate (ATP) production, and oxidative function associated with increased mitochondrial aging, depletion of substrate over time, and decrease in viability due to oxidative damage as more free radicals are produced by nonviable sperm over time. As expected STC increased over time in IVF media, due to increased cell death. In vivo however, the female genital fluids exert an influence on sperm motility such that some sperm that are immotile in vitro might regain motility in vivo and vice versa [79]. CASA-SCA clearly indicated within-individual differences confirming that a single spermatozoa fraction does not constitute a homogeneous fraction and also between-individual differences highlighting the need to select individuals carefully when performing AI or IVF. This study therefore enabled the characterization of motility patterns and associated with hyperactivation in antelope species, highlighting differences between species. Based on this study motility parameters such VSL, VAP, VCL, BCF, ALH, and RAP could be used to measure hyperactivity of these African antelope in vitro and may form an important step in understanding sperm physiology of the antelope and the development of species-specific IVF protocols for springbok, impala, and blesbok through assessment of motility characteristics, hyperactivation, and capacitation conditions. This information however should be treated carefully as minimum threshold settings for VSL, ALH, and a maximum threshold for LIN are required to identify hyperactivated sperm using CASA analysis systems [80]; however this was not in the scope of this study. The study does however provide preliminary data that can be used for defining these threshold values for particular events for the different antelope species. It is important to remember that because standard values have not been defined for normal or abnormal sperm, kinematic parameters for springbok, impala, and blesbok were obtained using a deer program, the results may need to be transformed to account for the factor of species differences between deer and the South African antelope in this study despite relatedness between the species. The CASA motility characteristics for springbok, impala, and blesbok as much as they provide valuable information about sperm physiology in these species should be considered with this in mind. Therefore, there is urgent need for CASA users to agree on standard analysis within a given species. However the information on the antelope motility characteristics gives preliminary reference data on these species and contributes to the development of

standardized values for these species. A comparison of CASA parameters between fresh and frozenthawed sperm may indicate the damage caused by cryopreservation/thawing and efficacy of the cryopreservation method used in this study. This study indicates differences in sperm physiology, physiological, and substrate requirements, and requirements for culture conditions between the species. This implies that optimal substrate concentrations and culture requirements for optimal function of antelope sperm from the different species is variable. This study has potential to address the gap in knowledge of antelope sperm research in terms of sperm physiology, comparative reproductive physiology of antelope and contributes towards protocol development of ex situ conservation of each species and therefore has implication on sperm fertility rate under in vitro conditions. 4.1. Conclusions Generally blesbok exhibited the lowest values of VCL, RAP, ALH, and BCF. Low values of VSL and elevated values of VCL and RAP could be used to measure hyperactivity of springbok, impala, and blesbok epididymal sperm in IVF media. Spermatozoa from the different antelope species have different physiological and optimal capacitation and culture requirements. The study contributes to comparative sperm physiology of the African antelope and the forms an important step in the development of species-specific IVF protocols for springbok, impala, and blesbok for ex situ conservation. Acknowledgments The following organizations and people are acknowledged for their contribution to this study: University of Johannesburg, Department of Zoology, Wildlife Biological Resources of National Zoological Gardens, National Research Foundation, Prof Steffens of STATCON, University of Johannesburg, Agricultural Research Council, Irene, Rebecca Krisher, Kim Labuschagne, Neil Mynhart, Zandile Matshazi, NECSA, Mazda Wildlife and Brakah Game Farms for assisting with sampling. References [1] Keith M, Knight MH, Castley GJ, Du Toit JT, Van Jaarsveld AS. The state of South Africa’s species. Johannesburg, South Africa: Endangered Wildlife Trust Conference; 2001. [2] Garde JJ, Soler AJ, Cassinello J, Crespo C, Malo AF, Espeso G, et al. Sperm cryopreservation in three species of endangered

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