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BOR - Papers in Press, published online ahead of print May 19, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026831
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BIOLOGY OF REPRODUCTION 71, 948–958 (2004)
DOI: 10.1095/biolreprod.103.026831
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Postthaw Evaluation of In Vitro Function of Epididymal Spermatozoa from Four Species of Free-Ranging African Bovids1

Jason R. Herrick3, Paul Bartels4, and Rebecca L. Krisher2,3

Department of Animal Sciences,3 Center for Comparative Medicine, Purdue University, West Lafayette, Indiana 47907 Wildlife Biological Resource Centre,4 Endangered Wildlife Trust, Pretoria, South Africa


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An improved understanding of reproductive physiology in nondomestic bovids is necessary for the development of assisted reproductive technologies (ARTs) for use in the conservation of endangered bovids. In this study, epididymal spermatozoa were recovered from blesbok (Damaliscus dorcas phillipsi), African buffalo (Syncerus caffer), springbok (Antidorcas marsupialis), and black wildebeest (Connochaetes gnou) following organized culls in South Africa. Our objectives were 1) to characterize the quality of epididymal spermatozoa, 2) evaluate the effectiveness of a cryopreservation protocol, and 3) compare postthaw sperm longevity (motility, viability, and acrosomal integrity) and functionality in two culture media with two capacitation reagents (caffeine and heparin). Following recovery, spermatozoa were diluted in EQ extender, slow-cooled, and frozen in the presence of 5% glycerol. Thawed spermatozoa were separated on a Percoll gradient and diluted in fertilization media (SOF for fertilization [SOFfert]; 0.6% BSA, 0.0 mM glucose, 25.0 mM NaHCO3) or modified SOFfert (1.2% BSA, 1.5 mM glucose, 37.0 mM NaHCO3) and either heparin or caffeine, and incubated for 6 h. Spermatozoa from these species maintained an average of 64% initial motility after thawing. Incubation medium and capacitation reagent had species-specific effects on the motility, viability, and acrosomal integrity of spermatozoa, suggesting ART procedures need to be optimized for each species. Springbok spermatozoa were also shown to be competent for in vitro fertilization. Information from this study concerning sperm physiology in blesbok, African buffalo, springbok, and black wildebeest will be useful in the development of ART for the conservation of these and other species of bovids.

assisted reproductive technology, gamete biology, in vitro fertilization, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Information regarding the reproductive physiology of many species of bovid is extremely limited compared to that for domestic cattle, sheep, goats, and, more recently, buffalo. The large gap existing between our understanding of reproduction in domestic and nondomestic bovids is largely due to the limited availability of nondomestic animals for research. However, several species of bovids are considered threatened or endangered, and an improved understanding of reproduction may facilitate efforts to conserve these species.

As the natural habitats for many species disappear, the remaining populations become isolated in small parks and reserves with little opportunity for genetic exchange between populations. As a result, genetic variability is diminished, and the viability of the population and the existence of the species is threatened. Similarly, maintenance of genetically viable populations in captivity, an extreme example of small, isolated populations, is an important component of species conservation [1]. Therefore, conservation efforts must focus on maintaining the genetic variability in isolated in situ and ex situ populations. Increasing our understanding of basic reproductive physiology will allow conservationists to monitor the reproductive health of populations and may facilitate efforts to maintain genetic diversity, including the development of assisted reproductive technologies (ARTs), in these small populations.

Assisted reproductive technologies such as artificial insemination (AI), in vitro fertilization (IVF), embryo transfer (ET), and gamete/embryo cryopreservation, are powerful tools that conservationists could use to manage and integrate wild and captive populations of endangered species [2, 3]. Implementation of ART allows exchange of genetic material between populations without transport of the animals, eliminates problems of behavioral incompatibility, overcomes physical conditions that limit breeding, and reduces opportunities for disease transmission [4, 5]. However, utilization of ART to its full potential is dependent on a thorough understanding of basic reproductive endocrinology and gamete/embryo biology [3, 5]. Recent research involving noninvasive (fecal and urinary) hormone monitoring [6, 7], sperm cryopreservation [8–10], and estrus synchronization [11] has expanded our understanding of reproduction in several bovid species, including the scimitar-horned oryx (Oryx dammah), sable antelope (Hippotragus niger), and Mohor gazelle (Gazella dama mhorr). In vitro fertilization has also been used to produce embryos from bongo (Tragelaphus euryceros) [12], addax (Addax nasomaculatus) [13], and gaur (Bos gaurus) [14], providing insight into the culture requirements for fertilization and the development of the preimplantation embryo. Although offspring have been produced through the use of AI or ET in a few species [9, 11, 14–16], success rates are low, and these techniques remain inefficient when compared to the success of similar techniques in domestic cattle.

One important component of AI or IVF/ET is viable, functional spermatozoa. When sperm collection is conducted at a different time or location (or both) from the female or oocyte collection, it is often necessary to cryopreserve the spermatozoa for storage, transport, or both. For AI, the functional life span of the spermatozoa after thawing dictates how closely insemination must be timed with ovulation. The lower the initial motility or viability and the shorter the longevity, the more important it is to tightly synchronize insemination and ovulation [17, 18]. One of the advantages of using AI is that spermatozoa are placed into the female reproductive tract, where conditions are optimal for sperm capacitation, fertilization, and embryonic development. In contrast, IVF requires the development of culture conditions that maintain sperm viability and motility, and support gamete interaction. However, IVF research also allows capacitation, oocyte penetration, and early embryonic development to be easily observed and manipulated, making it a useful technique for studying gamete physiology.

In the present study, epididymal spermatozoa were recovered from blesbok (Damaliscus dorcas phillipsi), African buffalo (Syncerus caffer), springbok (Antidorcas marsupialis), and black wildebeest (Connochaetes gnou) following organized culls and hunts in South Africa. Our first objective was to characterize the quality of epididymal spermatozoa and evaluate the effectiveness of a cryopreservation protocol developed for scimitar-horned oryx for use in these species. Second, we compared two culture media and two capacitation reagents (caffeine and heparin) for the ability to maintain in vitro motility, viability, and acrosomal integrity of the spermatozoa. Finally, springbok spermatozoa were coincubated with homologous oocytes to determine whether the cryopreservation procedure and culture conditions were supportive of IVF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Media Preparation

Unless specified otherwise, all chemicals were purchased from Sigma Chemical Company (St. Louis, MO). All media contained 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 ng/ml amphotericin (Gibco BRL Products, Gaithersburg, MD). Media were freshly prepared from stock solutions for each replicate, filtered (0.22 µm, Millex GV; Millipore Corp., Bedford, MA) immediately before use, and equilibrated at 39°C in the appropriate gas atmosphere.

Testes Collection and Gamete Recovery

Testes (three to four males per species) were recovered during the winter months (May through July) following managed culls or hunts within 4 h of death. Culls and hunts were conducted or supervised by professional hunting teams or the staff of the park (or both) and, to the best of our knowledge, none of the animals had been born captive or hand-raised. In all cases, animals were killed for reasons other than this study, and all tissue was collected opportunistically. Testes were maintained at 5 to 10°C for 2–8 h before dissection and sperm recovery. The epididymides and vasa deferentia were dissected from both testicles of individual males. The cauda epididymis and proximal vas deferens of each testicle was then flushed with 2 to 3 ml of isothermal Hepes-buffered SOF (SOF Hepes; 0.3% BSA, 1x nonessential amino acids [NEAA; ICN Biomedicals, Costa Mesa, CA], and 1.0 mM glutamine). Epididymal contents from both testicles of an individual male were pooled for processing.

Sperm Processing and Cryopreservation

Samples were centrifuged for 5 min to remove epididymal fluid, blood, or both, and diluted to 200–400 x 106 sperm/ml in EQ extender (20% egg yolk, 5.5% lactose, 1.5% glucose, and 0.25% Orvus Paste [Proctor and Gamble, Cincinnati, OH] [8, 19]). Extended sperm was placed in a water bath (20 to 25°C) and slowly (1.5 to 2 h) cooled to 4°C in a cold room. An equal volume of EQ containing 10% glycerol was then added in three steps (0.25x, 0.25x, and 0.5x original volume) at 20-min intervals, resulting in a 1:1 dilution of the original sample and final glycerol concentration of 5%. Samples with glycerol were equilibrated for an additional 1 h at 4°C before loading into straws. Straws were sealed and frozen by rapidly lowering them to the bottom of a dry shipper [8]. Straws were held for at least 10 min in the dry shipper before transfer to liquid nitrogen for storage.

Thawing and In Vitro Culture

Straws were briefly (10 sec) thawed in air before being plunged into a 38°C water bath for approximately 2 min. Thawed samples were layered onto a 45%:90% Percoll gradient [20, 21] and centrifuged for 25 min. The resulting pellet of live, motile sperm was resuspended in 5 ml of an SOF-based sperm washing medium [20] and centrifuged for 5 to 10 min. Final sperm pellets were diluted to 20–30 x 106 sperm/mL in either SOF for fertilization (SOFfert; 0.6% BSA, 0.0 mM glucose, 1.8 mM lactate, 25.1 mM NaHCO3; [20]) or modified SOFfert (modSOFfert; 1.2% BSA, 1.5 mM glucose, 23.7 mM lactate, 37.0 mM NaHCO3). Both media contained 1.7 mM CaCl2 and 2x NEAA and were further supplemented with penicillamine (10–20 µM), hypotaurine (5–10 µM), and either heparin (5 or 10 µg/ml) or caffeine (2.0 mM). Only one concentration (10 µg/ml) of heparin was tested on blesbok spermatozoa. Samples were incubated in gassed (5% CO2 in air) 1.5 ml microcentrifuge tubes at 39°C.

Evaluation of Motility, Viability, and Acrosomal Integrity

The percentage of motile spermatozoa (0% to 100%) and rate of forward progression (0, no movement to 5, rapid linear motion) were subjectively evaluated (100x) in at least four separate fields on a warm (37°C) slide immediately after recovery (before centrifugation and dilution in EQ); immediately after thawing (before Percoll processing); and 0, 3, and 6 h after Percoll separation and incubation. A sperm motility index (SMI; [percentage motile + {20 x rate}]/2) was calculated for each time point [22]. The efficiency of the cryopreservation procedure was determined by calculating the percentage of the initial SMI maintained after thawing ([postthaw SMI before Percoll/SMI after recovery] x 100). At each time point an aliquot (5–10 µl) of the sperm suspension was mixed (2:1, v/v) with an aliquot (2.5–5 µl) of stain containing Eosin B (2% w/v) and Fast Green FCF (0.8% w/v) in Lakes extender [23], smeared across a glass slide, and quickly dried with a hair dryer. Stained slides were assessed (200 sperm per treatment per time point) using brightfield microscopy (1000x) for the integrity of the plasma membrane (viability) and the acrosome. Spermatozoa containing a dark acrosome were considered to have a damaged acrosome, a result of cryopreservation or a spontaneous acrosome reaction, while spermatozoa with a clear or light-colored acrosome were considered intact. Spermatozoa with a clear or light-colored postequatorial segment were considered to be viable. Spermatozoa with a dark postequatorial segment were considered nonviable.

Evaluation of Epididymal Sperm Morphology

A small (15 µl) aliquot of the sperm suspension was diluted 1:1 (v/v) in 6.0% glutaraldehyde in PBS immediately after recovery from the testes and stored at 4°C until evaluated. An additional aliquot was fixed after thawing and processing through Percoll to determine the effectiveness of Percoll for eliminating abnormal spermatozoa. Morphological analysis was conducted using brightfield microscopy (1000x), and at least 200 spermatozoa were evaluated for each male at each time point [22].

Oocyte Collection and In Vitro Maturation

Springbok ovaries were also collected within 4 h of death as part of parallel studies of in vitro oocyte maturation. Ovaries were maintained in warm (approximately 30°C), 0.9% NaCl for 2–6 h before oocyte recovery. Ovaries were sliced repeatedly in warm (39°C) SOF Hepes to liberate cumulus-oocyte complexes (COCs). Oocytes completely surrounded by cumulus cells were selected, washed twice in SOF Hepes, and placed into one of two maturation media. One maturation medium was TCM-199 (Gibco BRL) containing 0.23 mM pyruvate, and 10% fetal bovine serum [20, 24]. The other medium used was Gmat [25], a defined medium based on G2 embryo culture medium, which contains 0.25 mg/ml hyaluronate, 0.5 mM citrate, and 0.5 mM cysteamine. Both media were supplemented with 0.01 U/ml bovine LH and FSH (Sioux Biochemicals, Sioux Center, IA), and 50 ng/ml epidermal growth factor. Oocytes were matured in 500 µl of medium in a four-well plate (Nunclon; Nalge Nunc International, Rochester, NY) or in 1.0 ml of medium in a cryovial (Nalge Nunc International) in an atmosphere of 5% CO2 in air at 39°C. After 22–24 h, COCs were transferred into drops of fertilization medium (see below) and maintained in 5% CO2 in air at 39°C until insemination.

In Vitro Fertilization

Springbok spermatozoa (2 x 106/ml) were coincubated with in vitro matured, homologous COCs in both fertilization media with both capacitation agents for 20 to 24 h in 5% CO2 in air at 39°C. Cumulus cells were removed by vortexing the COCs in 100 µl of SOF Hepes containing 0.01% (w/v; 80–160 U/ml) hyaluronidase for 3 to 4 min. Denuded zygotes were then compressed between a coverslip supported by a mixture of paraffin wax and petroleum jelly and a glass slide. Presumptive zygotes were then fixed in 3:1 ethanol:acetic acid for at least 24 h before staining with 1% (w/v) orcein in 45% (v/v) acetic acid and evaluating sperm penetration. Oocytes were considered penetrated or fertilized when at least two pronuclei were visible in the cytoplasm. When possible, meiotic stage of unfertilized oocytes was also noted.

Statistical Analysis

All percentage data were transformed (arcsin of the square root of the proportion) before analysis. Cryopreservation efficiency was analyzed using Proc GLM (Statistical Analysis Systems, Cary, NC) to determine differences between species. Following transformation, the proportions of normal sperm, sperm with cytoplasmic droplets, sperm with a bent midpiece, sperm with a bent or looped tail, or sperm with other abnormalities were analyzed using a paired t-test to compare initial morphology with morphology following thawing and Percoll processing. Motility (SMI), viability, and acrosomal status during culture were analyzed using Proc MIXED (Statistical Analysis Systems) with repeated measures [26]. The effects of base medium (SOFfert vs. modSOFfert), capacitation agent (capag; heparin vs. caffeine), and time, as well as all relevant interactions (base*capag, base*time, capag*time, and base*capag*time) were included in the model. Time was included as the repeated factor (0, 3, and 6 h of culture), and an unstructured covariance structure was used for repeated measures on each subject, designated as male(base*capag). When the two concentrations of heparin (5 and 10 µg/ml) were found to be not significantly different (P > 0.05), data were pooled for the final analysis. When a significant (P < 0.05) effect was found, a Fisher protected least significant difference test was used to determine treatment differences. Means were calculated prior to data transformation and are presented as ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Initial Sperm Parameters and Cryopreservation Efficiency

The lowest initial motility (46.3 ± 6.9 SMI) was found in springbok spermatozoa, with all other species having an initial SMI greater than or equal to 60.0 (Table 1). Across all species, 89.2% to 92.8% of spermatozoa were viable, and 92.4% to 96.6% of spermatozoa had an intact acrosome (Table 1). Following cryopreservation and thawing, SMI values ranged from 31.3 to 51.3 (Fig. 1). Cryopreservation efficiency was not different (P > 0.05) between species, with an average of 64.0% ± 3.1% (mean ± SEM) of initial SMI values maintained after thawing (blesbok, 58.6% ± 4.3%; buffalo, 60.0% ± 8.9%; springbok, 70.3% ± 10.0%; wildebeest, 68.4% ± 3.1%).


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  		TABLE 1. Initial sperm parameters



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  		FIG. 1. Initial (open bars) and postthaw (solid bars) sperm motility indices (SMI) for blesbok, buffalo, springbok, and black wildebeest spermatozoa. Percentages above postthaw values indicate the percentage of initial SMI recovered postthaw ([postthaw SMI/initial SMI] x 100)

Sperm Morphology Before and After Thawing and Percoll-Processing

Initially, less than 5.0% (1.3% to 3.6%) of spermatozoa were morphologically mature and normal (Table 2). In all species, the most prevalent morphology was the presence of cytoplasmic droplets (46.9% to 75.8%). Bent midpieces were also common (42.2% to 46.0%) in spermatozoa from buffalo, springbok, and wildebeest, but these occurred in less than 20% (15.9%) of spermatozoa from blesbok. Bent or looped tails and all other abnormalities were observed in less than 5% of evaluated spermatozoa. Following thawing and Percoll processing, there was a numeric increase in the proportion of normal spermatozoa in all species, but this increase was significant (P < 0.05) only in blesbok and springbok. In addition, there was a decrease in the proportion of spermatozoa with cytoplasmic droplets in all species, which was significant (P < 0.05) in blesbok. It was interesting to find that the proportion of spermatozoa with a bent or looped tail increased significantly (P < 0.05) in springbok, as did the proportion of other abnormalities. No differences were found in the proportions of spermatozoa with bent midpieces before or after thawing/Percoll processing in any of the four species.


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  		TABLE 2. Epididymal sperm morphology in blesbok, buffalo, springbok, and black wildebeest before and after Percoll processing

Effects of Culture Conditions on Sperm Motility, Viability, and Acrosomal Integrity

In blesbok, there was a significant decrease (P < 0.05) in motility, viability, and proportion of spermatozoa with intact acrosomes during the culture period (0 h>3 h>6 h; Fig. 2). Heparin (10 µg/ml) supported higher motility than caffeine (P < 0.05). There were significant (P < 0.05) interactions between time of culture and the base medium, as well as time, the base medium, and the capacitation agent. Therefore, the change in motility over time was affected by the base medium, and was further affected by the capacitation agent. The proportions of viable spermatozoa and the proportions of spermatozoa with intact acrosomes were not affected (P > 0.05) by base medium or capacitation agent, and none of the tested interactions were significant (P > 0.05).



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  		FIG. 2. Sperm motility index (SMI, A), viability (B), and acrosomal integrity (C) of blesbok spermatozoa cultured in SOFfert (F) or modSOFfert (MF) with heparin (H, 10 µg/ml) or caffeine (C, 2.0 mM). Summary (D) of the effects of time, base medium, and capacitation agent on sperm motility (SMI), viability, and acrosomal integrity. When significance (P < 0.05) or a statistical trend (P < 0.10) was found, the exact P value and the optimal treatment or treatments are shown. Nonsignificant (P > 0.05) effects are denoted by "NS." Solid bars, FH; diagonally striped bars, FC; open bars, MFH; checked bars, MFC

A difference (P < 0.05) between the effects of 5 and 10 µg/ml of heparin on buffalo spermatozoa was found only for motility (Fig. 3). Motility, viability, and the proportion of spermatozoa with intact acrosomes decreased (P < 0.05) from 0 to 3 h of culture, without a further, significant (P > 0.05) decline between 3 and 6 h of culture. Motility was best (P < 0.05) supported with 5 µg/ml heparin, with a significant (P < 0.05) interaction between capacitation agent and time of culture. In addition, there was a trend toward better (P = 0.08) motility in modSOFfert than in SOFfert. Viability was not affected (P > 0.05) by the base medium used, but there was a trend (P = 0.07) toward higher viability in the presence of heparin and an interaction (P = 0.05) between time and capacitation agent. Acrosomal integrity was not affected (P > 0.05) by base medium or capacitation agent, but the interactions between time and capacitation agent and time, base, and capacitation agent were significant (P < 0.05).



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  		FIG. 3. Sperm motility index (SMI, A), viability (B), and acrosomal integrity (C) of buffalo spermatozoa cultured in SOFfert (F) or modSOFfert (MF) with heparin (H, 5–10 µg/ml) or caffeine (C, 2.0 mM). Summary (D) of the effects of time, base medium, and capacitation agent on sperm motility (SMI), viability, and acrosomal integrity. When significance (P < 0.05) or a statistical trend (P < 0.10) was found, the exact P value and the optimal treatment or treatments are shown. Nonsignificant (P > 0.05) effects are denoted by "NS." Light gray bars, F5H (A), FH (B, C); diagonally striped bars, F10H (A), FC (B, C); open bars, FC (A), MFH (B, C); checked bars, MF5H (A), MFC (B, C); dark gray bars, MF10H (A); horizontally striped bars, MFC (A)

There was no difference (P > 0.05) between the effects of 5 or 10 µg/ml heparin on the motility, viability, or acrosomal integrity of springbok spermatozoa, so data were pooled for analysis (Fig. 4). Time had a significant effect (P > 0.05) on all end points. Motility decreased significantly (P < 0.05) between each time point (0>3>6 h). Motility was higher (P < 0.05) when spermatozoa were cultured in modSOFfert than in SOFfert, and tended (P = 0.10) to be higher in caffeine than in the presence of heparin. There was also a significant (P < 0.05) base medium by capacitation agent interaction in which the highest motility was observed after culture in modSOFfert with caffeine. In addition, there was an interaction (P = 0.05) between time of culture and capacitation agent. Viability and acrosomal integrity decreased (P < 0.05) from 0 to 3 h of culture, but they did not decrease (P > 0.05) between 3 and 6 h of culture. There was a trend (P = 0.08) toward higher viability in the presence of heparin than caffeine, as well as a significant (P < 0.05) interaction between time of culture, base medium, and capacitation agent. Base medium, capacitation agent, and all tested interactions did not significantly (P > 0.05) affect the acrosomal status of springbok spermatozoa.



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  		FIG. 4. Sperm motility index (SMI, A), viability (B), and acrosomal integrity (C) of springbok spermatozoa cultured in SOFfert (F) or modSOFfert (MF) with heparin (H, 5–10 µg/ml) or caffeine (C, 2.0 mM). Summary (D) of the effects of time, base medium, and capacitation agent on sperm motility (SMI), viability, and acrosomal integrity. When significance (P < 0.05) or a statistical trend (P < 0.10) was found, the exact P value and the optimal treatment or treatments are shown. Nonsignificant (P > 0.05) effects are denoted by "NS." Solid bars, FH; diagonally striped bars, FC; open bars, MFH; checked bars, MFC

For black wildebeest spermatozoa, there was a significant difference (P < 0.05) between the effects of 5 and 10 µg/ml heparin on motility and viability, but no difference (P > 0.05) for the effects on acrosomal status (Fig. 5). Motility, viability, and the proportion of spermatozoa with intact acrosomes decreased significantly (P < 0.05) from 0 to 3 h of culture. There was also a decrease (P < 0.05) in viability between 3 and 6 h of culture, but only a trend for a decrease in motility (P = 0.05) and the proportion of spermatozoa with intact acrosomes (P = 0.09) during this period. Motility of wildebeest spermatozoa was significantly affected (P < 0.05) by capacitation agent, with the highest motility in the presence of 5 µg/ml heparin or caffeine. There was also a trend (P = 0.08) toward better motility when spermatozoa were cultured in modSOFfert. Viability was higher (P < 0.05) in the presence of 5 µg/ ml heparin than 10 µg/ml heparin or caffeine, but a higher proportion of spermatozoa (P = 0.09) had intact acrosomes in the presence of caffeine than heparin. Base medium did not affect (P > 0.05) viability or acrosomal integrity. Finally, there was a significant (P < 0.05) interaction between time and the capacitation agent used on the acrosomal status of wildebeest spermatozoa, but there were no other significant (P > 0.05) interactions between main effects on motility, viability, or acrosomal integrity.



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  		FIG. 5. Sperm motility index (SMI, A), viability (B), and acrosomal integrity (C) of black wildebeest spermatozoa cultured in SOFfert (F) or modSOFfert (MF) with heparin (H, 5–10 µg/ml) or caffeine (C, 2.0 mM). Summary (D) of the effects of time, base medium, and capacitation agent on sperm motility (SMI), viability, and acrosomal integrity. When significance (P < 0.05) or a statistical trend (P < 0.10) was found, the exact P value and the optimal treatment or treatments are shown. Nonsignificant (P > 0.05) effects are denoted by "NS." Light gray bars, F5H (A, B), FH (C); diagonally striped bars, F10H (A, B), FC (C); open bars, FC (A, B), MFH (C); checked bars, MF5H (A, B), MFC (C); dark gray bars, MF10H (A, B); horizontally striped bars, MFC (A, B)

In Vitro Fertilization

Of 132 in vitro matured springbok oocytes inseminated and fixed for evaluation of sperm penetration, only four oocytes had two or more pronuclei and were classified as penetrated (Table 3; Fig. 6). Meiotic stage was assessed in 45 of 120 oocytes (37.5%) matured in Gmat, with between 0% and 42% of oocytes reaching metaphase II across replicates, or an overall mean of 26.7% (data not shown). All four penetrated oocytes were found in oocytes that were matured in TCM-199 with 10% serum and inseminated in modSOFfert supplemented with caffeine.


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  		TABLE 3. Results of in vitro fertilization of in vitro matured (22 to 24 h) springbok oocytes with cryopreserved, epididymal spermatozoa



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  		FIG. 6. Fertilized springbok oocyte showing two pronuclei


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the amount of knowledge available concerning the reproductive physiology of nondomestic species has expanded greatly in recent years, there are still many species for which available data are extremely limited and many more species that have yet to be studied at all. For the species in this study, with the possible exception of the African buffalo, only a small number of reports exist concerning sperm physiology, cryopreservation, or both [27– 32]. The results of this study indicate that high-quality (motile and viable with an intact acrosome) epididymal spermatozoa can be recovered from blesbok, African buffalo, springbok, and black wildebeest. On average, approximately 60% to 70% of initial, prefreeze motility can be recovered after cryopreservation with a "field-friendly" protocol developed for the scimitar-horned oryx. After isolation of motile sperm using a Percoll gradient, viable, motile spermatozoa from these species could be maintained in culture for up to 6 h. Finally, cryopreserved epididymal spermatozoa from springbok were competent to fertilize in vitro matured homologous oocytes.

Sperm cryopreservation is a critical component of many ARTs because it allows for long-term storage and transport of sperm. Once frozen, the sperm can be used for research purposes or for the production of embryos, offspring, or both following AI or IVF and ET. Recently, Roth et al. [8] developed a cryopreservation protocol for scimitar-horned oryx that was subsequently shown to be useful for fringe-eared oryx [33]. An important component of this technique is that it utilizes a single-step freezing method in which the straws containing sperm are rapidly lowered into a dry shipper. Because this does not require liquid nitrogen vapor or dry ice, it has been proposed that this method is well-suited for work conducted in the field. In addition to scimitar-horned oryx and fringe-eared oryx, this procedure was also shown to be suitable for spermatozoa from the eland, blesbok, black wildebeest, and buffalo in a preliminary study [32]. In the present study, the suitability of this cryopreservation protocol for a variety of species was further demonstrated. Approximately 64% of prefreeze SMI was maintained after thawing in all species examined, and there was no difference between species. Finally, sperm cryopreserved using this technique have been shown to be competent to penetrate homologous (springbok, present study) and heterologous (domestic cattle) oocytes in vitro [8, 17, 33]. Based on the previous work with spermatozoa from scimitar-horned oryx, fringe-eared oryx, and Sumatran and black rhinos [34], as well as the present study, cryopreservation of spermatozoa in EQ with glycerol using the single-step dry shipper method appears to have broad applicability to a variety of species.

Another important finding of this study was the species-specific preferences for culture media and capacitation agents between the four species. While modSOFfert proved optimal for sperm motility in springbok, buffalo (statistical trend), and black wildebeest (statistical trend), there was no effect of base medium on motility of blesbok spermatozoa. Similarly, motility of blesbok, buffalo, and black wildebeest spermatozoa was highest in the presence of heparin, while spermatozoa from springbok maintained better motility in the presence of caffeine. Significant interactions between culture conditions (base medium, capacitation agent, or both) and time on sperm motility, viability, and acrosomal status, further demonstrate that culture conditions affect sperm physiology. Similarly, previous work on the scimitar-horned oryx and fringe-eared oryx has indicated species differences in sperm function and fertilization mechanisms between closely related (Oryx genus) bovid species [33, 35]. These findings have important implications for the development of ART. Due to the limited amount of information concerning most nondomestic species, preliminary research concerning ART often use protocols developed for domestic species, primarily cattle, as a starting point [12– 14, 31, 33, 35]. It was interesting that SOFfert and heparin, the treatments used in our laboratory for domestic cattle [20], were not optimal for springbok spermatozoa. Perhaps other domestic bovids, such as sheep or goats, could provide more appropriate models for preliminary research in antelope.

The modSOFfert was designed with higher concentrations of bicarbonate, BSA, lactate, and glucose than those found in SOFfert. Bicarbonate is found at high concentrations (30 to 90 mM) within the oviduct and is known to play a prominent role in the control of sperm motility, capacitation, and oocyte penetration [36–39]. The concentration of bicarbonate also determines the pH of the culture media when incubated with elevated (5% to 10% in air) CO2 [40]. In pigs and mice, the beneficial effects of bicarbonate on capacitation are independent of the effects on pH, but it is not clear how pH affects other aspects of sperm physiology [39, 41]. The possible effects of bicarbonate ions on sperm motility, sperm capacitation, extracellular pH, or a combination of these may account for the beneficial effects of modSOFfert on springbok sperm motility and oocyte penetration.

Blood-derived products such as serum and BSA are commonly used macromolecules in culture media, mimicking the presence of albumin and other proteins in the oviduct [40, 42, 43]. In fertilization media, serum and BSA promote the efflux of cholesterol from the sperm membrane [44, 45]. By altering the cholesterol content, and therefore the fluidity, of the sperm membrane, protein in the media plays a critical role in the initiation of capacitation. In support of this, increasing concentrations of BSA lead to increased oocyte penetration in pigs [46] and alternative cholesterol acceptors (cyclodextrans) can mimic the effects of BSA on capacitation [47, 48]. In addition, BSA is known to be contaminated with various substances including citrate, lactate, and fatty acids that may alter sperm physiology [49]. The higher (12 mg/ml) concentration of BSA in modSOFfert may have contributed to the higher motility of springbok, buffalo, and black wildebeest spermatozoa, as well as successful oocyte penetration in springbok, in this medium.

Carbohydrates are another important group of culture media components that influence both the metabolism and viability of gametes and embryos. In spermatozoa, glucose serves as an energy source and participates in sperm motility, capacitation, the acrosome reaction, and sperm-oocyte fusion [50–52]. In cattle spermatozoa, the effects of glucose on capacitation are contradictory. Parrish et al. [53, 54] found that heparin-induced sperm capacitation is delayed when glucose is present in the medium, and that this delay is related to glucose metabolism and intracellular pH. However, successful bovine sperm capacitation and IVF have also been reported in the presence of high (14.0 mM) concentrations of glucose in a variety of media in which penicillamine, caffeine, or both were used to capacitate the spermatozoa in the presence of elevated bicarbonate [55, 56]. Parrish et al. [54] attributed the inhibitory effects of glucose to a delay in intracellular alkalinization, so an excess of bicarbonate may have been able to overcome this effect. Lactate participates in the control of metabolism and the maintenance of intracellular pH and REDOX state in oocytes and embryos, and may have similar effects in spermatozoa [57, 58]. Media containing concentrations of D,L-lactate greater than or equal to 20.0 mM support capacitation, fertilization, or both in bull [53], goat [59], and mouse [60] spermatozoa. Based on these findings, glucose (1.5 mM) and lactate (D,L-lactate 23.7 mM) were included in modSOFfert and may have contributed to the positive effects of modSOFfert.

In many species, IVF media contain chemical agents, such as heparin or caffeine, to promote sperm capacitation. Heparin is a common addition to bovine IVF medium, largely based on the findings of Parrish et al. [53]. This glycosaminoglycan is similar to a capacitation-inducing component from estrual cow oviductal fluid and stimulates capacitation by increasing intracellular cAMP and Ca+2 concentrations [61–63]. Caffeine is a phosphodiesterase inhibitor, also leading to increased intracellular cAMP, and is routinely used to promote capacitation in porcine and primate sperm [46, 64]. As with most culture components, numerous interactions can exist between heparin or caffeine and other substances present during IVF. As mentioned above, significant interactions between glucose and heparin, and possibly bicarbonate, exist during capacitation of bovine sperm [53, 54]. In addition, synergistic effects of caffeine and bicarbonate are known to alter the ability of porcine sperm to penetrate oocytes [41]. In the present study, the time by capacitation agent effect on the proportion of sperm with an intact acrosome was significant in buffalo and black wildebeest. In all species, the progressive decrease in acrosomal integrity over time was likely affected by the simultaneous loss of viability and the associated loss of membrane integrity. Further work involving chemical-induced, zona pellucida-induced (or both) acrosome reactions would be useful in determining whether the effects seen in our study were affected more by changes in viability or the incidence of spontaneous acrosome reactions in capacitated spermatozoa.

Another key finding in the present study was the successful IVF of springbok oocytes. To our knowledge, this is the first report of successful IVF in springbok, and the first using in vitro matured oocytes and cryopreserved epididymal spermatozoa. This finding provides further validation for the effectiveness of the cryopreservation protocol for springbok epididymal spermatozoa, which exhibited the lowest initial and postthaw motility of the species studied here. In addition, successful IVF demonstrates the potential for embryo production following postmortem gamete collection. However, our results only illustrate the feasibility of such technology and, more importantly, highlight the need for further optimization of these procedures. For example, despite the use of conditions known to support sperm motility and viability (modSOFfert with caffeine), fertilization success was low (4 of 41 oocytes matured in either Gmat or TCM199).

Two critical factors for IVF success that may have influenced our findings, yet were not addressed in this study, are the timing of oocyte maturation and the effects of season. Although 27% of oocytes had reached metaphase II at the time of evaluation (approximately 22 h postinsemination or 44 h postmaturation), it could not be determined how long these oocytes had been at that stage of meiosis. Studies in addax have shown that maturation is delayed relative to domestic species [65]. In addition, a recent study from our laboratory found that springbok oocytes also matured later (28 h) following in vitro maturation in Gmat [66]. It is interesting that the proportions of oocytes found to be at metaphase II 20–24 h after the initiation of culture in that study was similar to our findings, perhaps suggesting that meiosis did not progress after COCs were removed from maturation medium and inseminated. Similarly, the improved IVF success rate using TCM-199 for oocyte maturation might indicate that the kinetics of oocyte maturation are different from what was observed in Gmat. Alternatively, the improved motility of springbok spermatozoa in modSOFfert with caffeine may have allowed more spermatozoa to be motile at the time oocytes reached metaphase II, approximately 4–8 h after insemination [66]. Synchronizing the time oocytes reach metaphase II and the time of insemination may improve fertilization rates. Another contributing factor may have been seasonality. Oocytes and spermatozoa were collected during the winter months in South Africa (May through July), a time when seminiferous tubule diameter, testes weight, sperm number, and sperm motility are at their lowest levels in springbok [67]. The same is true for blesbok, impala, kudu, black wildebeest, and red hartebeest [67, 68]. This time of year was chosen because it coincides with the time of most hunting and culling practices. Corresponding studies during the spring or summer would be of interest to determine whether seasonality affects gamete quality, as shown in other species [69].

As expected for epididymal spermatozoa, a high proportion of immature (cytoplasmic droplets), abnormal (bent midpieces), or both sperm forms were seen in all of the species studied [70–73]. Spermatozoa with cytoplasmic droplets are common among epididymal spermatozoa, because this is a normal stage in sperm maturation [70, 71]. Consequently, this is a common morphology among epididymal spermatozoa collected from domestic pigs [72, 73] and cats [74], as well as springbok, blesbok, and impala [29]. Although the presence of these forms in the epididymis is likely a normal phenomenon, the proportion of these forms may vary with season, as demonstrated for sperm motility from many of these same species [67, 68]. For example, seasonal variation in the proportion of normal sperm and the incidence of bent midpieces and retained cytoplasmic droplets has been reported for goats, cattle, and African buffalo [75–77]. Although Percoll processing statistically improved sperm morphology, the proportion of normal spermatozoa following Percoll was less than or equal to 32% across species. Therefore, the use of Percoll may be more effective for improving motility rather than the morphology of epididymal spermatozoa. It is interesting that the proportion of springbok sperm with bent or looped tails, as well as spermatozoa with other abnormalities, increased following thawing and Percoll processing. It has been reported that tail abnormalities can be induced by osmotic shock or temperature shock [72], suggesting that springbok sperm may be more sensitive to the cryopreservation process than the other species. In support of this, Loskutoff et al. [29] found that springbok spermatozoa were less resistant to cooling in the absence of an extender than sperm from blesbok or impala. In addition, initial sperm morphology has been related to the resistance of feline sperm to temperature and osmotic fluctuations [78, 79]. Further research concerning the cryosensitivity of springbok spermatozoa, as well as the interactions between sperm morphology, season, and cryotolerance, would be interesting in determining the etiology of these effects.

In conclusion, viable, motile spermatozoa can be recovered postmortem from blesbok, buffalo, springbok, and black wildebeest and successfully cryopreserved in EQ using a single-step freezing method. Following thawing, approximately 64% of initial motility can be recovered. Further isolation of motile spermatozoa using a Percoll gradient results in motile sperm with improved morphology that can maintain motility and viability for up to 6 h of culture under IVF conditions. Incubation medium and capacitation reagent were found to have species-specific effects on the motility, viability, and acrosomal integrity of spermatozoa following cryopreservation. These findings suggest that procedures for IVF need to be optimized for each species to identify conditions that support motility, as well as sperm capacitation and oocyte penetration. In addition, parallel studies of oocyte physiology are necessary to improve the development of IVF procedures. This study provides important initial information concerning sperm physiology in blesbok, African buffalo, springbok, and black wildebeest that will be useful in the development of ART for the conservation of these and other species of bovids.


   ACKNOWLEDGMENTS
 
We thank all the parks and reserves, as well as the hunting teams, for allowing us to collect gonadal tissue. We are also grateful to Isabel Boshoff, Brenda Daly, Belinda Spillings, and numerous volunteers at the Wildlife Biological Resource Centre for technical assistance. Dr. Normand St.-Pierre is thanked for his help with the statistical analysis and Dr. Wayne Singleton is thanked for his comments on an early version of this paper.


   FOOTNOTES
 
1 Travel expenses for J.H. were provided through the W.R. Featherston Off-Campus Training Fellowship (Department of Animal Sciences, Purdue University) and a Development Travelling Fellowship (Company of Biologists, Ltd.). Back

2 Correspondence: Dr. Rebecca L. Krisher, Department of Animal Sciences, Lilly Hall of Life Sciences, 915 W. State Street, Purdue University, West Lafayette, IN 47907. FAX: 765 494 9346; rkrisher{at}purdue.edu Back

Received: 19 December 2003.

First decision: 13 January 2004.

Accepted: 12 May 2004.


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