Tris-citric Egg Yolk Research Articles

Testosterone secretion, testicular histology and the cryopreservation of cauda epididymidal spermatozoa in the common ringtail possum (Pseudocheirus peregrinus)

The present study reports novel aspects of the reproductive biology of the male common ringtail possum (Pseudocheirus peregrinus). Plasma testosterone was measured through a stimulation test using the gonadotrophin-releasing hormone agonist, buserelin. Following intra-muscular administration of buserelin, there was an increase (P<0.05) in testosterone concentration in the peripheral circulation 4 h later. Quantitative testicular histology of this species was described for the first time. Eight stages of the seminiferous epithelium cycle were identified in 10 possums and their relative frequency determined. Spermatozoa were recovered from the cauda epididymides of hemi-castrated possums and cryopreservation conducted in straws (6 degrees C min(-1)) using final glycerol concentrations ranging between 2 and 20% in Tris-citrate egg yolk extender (v/v). Frozen straws were thawed and post-thaw motility, rate of motility, the percentage of live-dead spermatozoa and the percentage of sperm with swollen decondensed nuclei recorded. Similar to other marsupial sperm, common ringtail possum cauda epididymidal spermatozoa required high levels of glycerol (10-16%) in order to maintain post-thaw viability.

The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.

Modulation of post-thaw sperm functions with oviductal proteins in buffaloes

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 μm) and frozen at −20 °C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at −20 °C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris–egg yolk–citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN 2 (−196 °C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1 mg/ml of extended semen. The equilibrated and frozen-thawed (37 °C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 °C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher ( P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher ( P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher ( P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher ( P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.

214USE OF FLOURESCENT PROBES TO ACCESS EPIDIDYMAL SPERMATOZOA OF THE BLUE WILDEBEEST CONNOCHAETES TAURINUS AND IMPALA ANTELOPE AEPYCEROS MELAMPUS MELAMPUS

Sperm quality assessment may be a useful tool not only for evaluating the reproductive health of free-ranging populations, but also for selecting individuals for future assisted-reproduction technology programs. The aim of this study was to assess the functionality of epididymal spermatozoa collected from blue wildebeest (Connochaetes taurinus) and impala (Aepyceros melampus melampus) during the non-breeding season, using the fluorescent probes, propidium ioide (PI;; Sigma, South Africa) and JC-1 (Molecular Probes, The Netherlands). Six blue wildebeest and eight impala were harvested as part of a wildlife management program on a game ranch in South Africa. Testes were removed and transported to the laboratory within 6 hours while being maintained at 4°C. The cauda epididymides were removed and flushed with 1mL of Tris-citrate egg yolk extender (fraction A, Biladyl;; Minitüb, Germany). The sperm sample was diluted 1:4 in HEPES washing medium (Sigma;; 20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH;; 400mOsm/kg, pH 7), and centrifuged for 5min at 600g, followed by re-suspending the pellet in 0.1mL of HEPES saline medium (Sigma;; as for washing medium, except 197mM NaCl instead of sucrose). The percentage of motile (MS) and progressively motile (PS) spermatozoa were determined using phase contrast microscopy (×200, 37°C). Sperm plasma membrane integrity and mitochondrial status were assessed using fluorescence microscopy (×400, 450–490nm excitation filter, 510nm dichroic-beam splitter, 520nm barrier filter) after staining with PI (50ngmL−1; 10min, RT) and JC-1 (7.5μM; 30min, 37°C), respectively. Spermatozoa with damaged plasma membranes showed a red fluorescence and spermatozoa with active and inactive mitochondria (MIT) fluoresced orange and green, respectively. Spearman correlation coefficients were calculated between spermatozoa with intact plasma membranes (IPM) and MIT, and with motility (Statistica™ package). A summary of the results is shown in the table 1. Although samples were not collected during the breeding season, sperm quality appeared to be good for the blue wildebeest, but less so for the impala. In general, impala results were more varied. Significant correlations were found for impala (n=8, P&amp;lt;0.05) MS-IPM: 0.75; IPM-MIT: 0.83, and for blue wildebeest (n=6, P&amp;lt;0.05), MS-IPM: 0.84; IPM-MIT: 0.81, and for pooled data (n=14, P&amp;lt;0.01), MS-IPM: 0.93; MS-MIT: 0.87; PS-IPM: 0.67; PS-MIT: 0.66; IPM-MIT: 0.95. These correlations suggest a relationship of functional parameters to sperm motility. Both membrane integrity and mitochondrial status are important for sperm flagellar activity. The correlation between IPM and MIT indicates a relationship or the effect of common factors. In conclusion, sperm collected from blue wildebeest and impala during the non-breeding season appear functional, a fact that may be useful for future conservation programs based on assisted reproduction technology or for assessing the reproductive health status of free-ranging wildlife populations. The fluorescent probes PI and JC-1 appear useful for assessing sperm quality in these two species and should be considered for further sperm quality assessment studies in other antelope species. Table 1 Results of the analyses, showing mean±SD (max.–min.)

208EFFECT OF REFRIGERATION AND CRYOPRESERVATION ON THE QUALITY OF LION EPIDIDYMAL SPERMATOZOA

Epididymal spermatozoa from harvested wild animals is potentially useful for conservation purposes, as it can be used for subsequent artificial insemination or stored in Biological Resource Banks for future use. The potential of sperm banking is of particular interest for use in lion (Panthera leo) populations maintained in small National Parks, as translocation of males to effect gene-flow is often problematic, resulting in the translocated lion being killed by resident pride males. We measured the change in sperm quality over time during cool storage (at 4°C) and after thawing of samples cryopreserved at −196°C. Also, we present a correlation between sperm plasma membrane integrity and mitochondrial activity as measured by fluorescent analysis. The testes from a pride lion were removed and transported to the laboratory (at 4°C) within 6h. The epididymides were removed and both cauda epididymides were flushed with 1mL of Tris-citrate egg yolk extender (Fraction A, Biladyl, Minitub, Germany). The sample containing 2930×106 cells mL−1 was washed (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH;; 400mOsm/kg, pH 7; Sigma, South Africa) and after centrifugation (5min. at 600g), the pellet was resuspended in 0.5mL of washing solution (with 197mM NaCl instead of sucrose). One aliquot of spermatozoa was kept at 4°C and evaluated at 24h intervals for 7 days. A second aliquot of the sperm sample was extended in Tris-citrate egg yolk extender with glycerol (Fraction B, Biladyl), frozen in liquid nitrogen (LN) vapor and stored in LN. The frozen sample was later thawed and evaluated as for the cooled samples. Percentages of motile (MS) and progressive (PS) spermatozoa were assessed using a phase contrast microscope (×200; stage at 37°C). Sperm plasma membrane damage was assessed by determining the percentage of cells exhibiting red fluoresence after staining with propidium iodide (PI, 50ng/mL; 10min RT). Spermatozoa that did not stain red in PI were classified as plasma membrane intact (PMI). Resilience to hypo-osmotic shock and plasma membrane integrity were evaluated by incubating a portion of the sample in a 100mOsm/kg solution (10nM glucose, 20nM HEPES, 30nM NaCl) containing PI for 15min at room temperature. The percentage of sperm cells with active mitochondria (MIT) was determined by counting spermatozoa showing orange fluoresence over the mid-piece after staining with JC-1(7.5 uM Sigma) for 30min at 37°C. At collection, MS was 15% and did not show a significant decrease during the 7-day storage period. Initially, PS was 10% and dropped to 5% after 7 days, with values fluctuating during the storage period. Both PMI and HOSPMI were 80% on Day 1, gradually decreasing to 75% on Day 7 of storage. PMI and MIT showed a highly significant correlation (r=0.88; P=0.003; n=8). In frozen-thawed sperm samples, MS fell from a pre-freeze value of 15% to 5% after thawing. Similarly, PS fell from 10% in pre-freeze to 3% in frozen-thawed samples. Likewise, PMI, HOSPMI and MIT values were 80% and 45%, 87% and 45% and 89% and 49%, respectively. Our study showed that lion sperm PMI and MIT remained high after 7 days at 4°C. MS and PS, although low, did not vary during this same period. PI and JC-1 showed a significant correlation, suggesting that both might be affected by the same deleterious factors. Although PMI, HOSPMI and MIT values decreased approximately 40% after freezing, we feel that such sperm samples could be used for in vitro embryo production, if not by IVF, by ICSI. Of course, additional studies are needed to validate our suggestion.

211QUALITY CHANGES IN BLUE WILDEBEEST (CONNOCHAETES TAURINUS) EPIDIDYMAL SPERMATOZOA MAINTAINED AT 4°C

Wildlife management in southern Africa often involves the harvesting of animals on ranches and reserves, providing unique opportunities to collect and assess the quality of epididymal spermatozoa for possible future conservation actions. The black wildebeest (Connochaetes gnu) is facing renewed threats to its survival, including the production of fertile hybrids from crossing with the more common blue wildebeest (Connochaetes taurinus). The close relationship between the two wildebeest species allows for the blue wildebeest to be used as a model to assess epididymal sperm quality over time while maintained at 4°C. Field conditions often preclude the immediate availability of liquid nitrogen, necessitating the development of alternative short-term storage methods. All chemicals were provided by Sigma (South Africa) unless otherwise stated. Testes were harvested from 6 blue wildebeest bulls at a local game farm, Savannah, and kept at 5°C during transportation to the lab. Epididymides were dissected out and spermatozoa were flushed out of the cauda epididymis using 1mL of Tris-citrate egg yolk extender (Fraction A, Biladyl, Minitub, Germany), followed by storage at 4°C and assessment at 12h intervals. At each interval, an aliquot was removed, washed with a modified buffered HEPES solution (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH;; 400mOsm/kg, pH 7; Sigma) and visually assessed with a phase contrast microscope (×200, at 37°C) to determine the percentage of motile (MS) and progressive motile (PS) spermatozoa. In addition, plasma membrane integrity (PMI) was assessed with eosin-nigrosin staining and active mitochondrial status (MIT) assessed with an epifluorescent microscope (×400) using the fluorescent probe JC-1 (Molecular Probes, The Netherlands;; 7.5μM; 30min at 37°C). Resilience to hypo-osmotic shock was also evaluated by incubating the sample in a modified hypo-osmotic medium (100mOsmkg−1; 15min RT), and staining with PI to assess plasma membrane integrity (HOSPMI). A summary of results is presented in the table 1. The MS, MIT and HOSPMI did not decrease significantly during the 48h storage period. The only parameters that showed a significant decrease were PS and PMI (P&amp;lt;0.01, Kruskall-Wallis test). However, PMI showed a slow but steady decrease (13%), whereas PS underwent a significant drop (52%). In conclusion, epididymal spermatozoa from the blue wildebeest, kept at 4°C for 48h, may still be useful for some assisted-reproduction techniques. The use of spermatozoa from a common but closely related wildebeest species allows for the development of assisted-reproduction techniques that may one day aid the conservation of threatened wildebeest species. Additional research is needed to confirm these findings and to test the effect of longer storage times on spermatozoa of this species as well as closely related endangered species. Table 1 Parameters measured during the 12-h time periods (mean±SD)

209FUNCTIONAL ASSESSMENT OF WHITE RHINOCEROS CERATHOTERIUM SIMUM EPIDIDYMAL SPERMATOZOA BEFORE AND AFTER CRYOPRESERVATION

Biological Resource Banks represent a potentially valuable tool for species conservation. It is, however, necessary to understand the species-specific cryopreservation process and its consequences for spermatozoa to aid in the development of assisted reproduction as a future conservation tool. The aim of this study was to assess the in vitro functionality of white rhinoceros Cerathoterium simum epididymal spermatozoa both before and after cryopreservation. Testes from a harvested white rhino bull were removed and transported at 5°C to the laboratory within 4h. The cauda epididymis was dissected out and flushed with 2mL of Tris-citrate egg yolk extender (fraction A, Biladyl, Minitüb, Germany). A 0.1mL aliquot was removed for analysis and the balance (9mL; 2mL fraction A+7mL sperm sample) mixed with an additional 27.2mL of Tris-citrate egg yolk with glycerol (fraction B, Bidadyl). The extended sample was allowed to cool to 4°C over a 6-h period before an additional 29.2mL of cooled fraction B were added (final sperm concentration=150×106mL−1). Sperm samples were loaded into 0.25-mL straws and frozen over LN2 vapor (4cm for 20min) for later assessment. Sperm straws were thawed by placing the straws in water at 37°C for 30s. Pre-freeze and post-thaw evaluations were carried out in the same manner. Media used included: HEPES for washing (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH) and HEPES saline (197mM NaCl, instead of sucrose). An aliquot was diluted with HEPES (washing) and centrifuged for 5min at 600×g; the pellet was resuspended in HEPES saline. Sperm motility (total motility %, TM;; and progressive motility %, PM) was assessed using phase contrast microscopy (×200; 37°C). Sperm plasma membrane status was assessed using the fluorescent dye, propidium iodide (50ngmL−1 in HEPES saline;; 10min, RT). Percentage of cells with plasma membranes intact (unstained;; PMI) was recorded. Mitochondrial status was assessed with the fluorescent dye, JC-1 (7.5μM in HEPES saline;; 30min, 37°C). The % of cells with an orange-stained midpiece was recorded (active mitochondria;; MIT). Resilience to hypoosmotic shock (HOS test) was assessed by diluting a sample in 100mOsm/kg HEPES saline (1:20; 15min, RT). An aliquot was stained with PI to assess plasma membrane status (HOSPMI), and the rest was fixed with formaldehyde, and % coiled tails (positive endosmosis;; HOST) was estimated using phase contrast microscopy (×400). Evaluations of PMI, MIT and HOSPMI were performed using fluorescence microscopy (×400, 450–490nm excitation filter). The results indicated that quality was good pre-freezing (TM: 60%; PMI: 86%; MIT: 100%), except for a PM value of 15%. After thawing, although there was a drop in TM (30%), there was no decrease in PM (20%). Our in vitro functional assessment indicated a loss of quality between the pre-freeze and post-thaw evaluations, but PMI and MIT maintained their pre-thaw levels (60% and 72%, respectively). The HOS test, which indicates plasma membrane integrity, decreased from the pre-freeze level (91%) to a post-thaw value of 70%. HOSTPMI was 72% pre-freeze, and decreased to 54% post-thaw. In conclusion, epididymal spermatozoa from the white rhino may retain its functionality after cryopreservation in a commerically available cryo-extender (Bidadyl). The use of assisted reproduction techniques could someday play a role in the management and conservation of the white rhinoceros and related species.