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

2004 ◽  
Vol 16 (2) ◽  
pp. 228
Author(s):  
T. Spies ◽  
F. Olivier ◽  
F. Martinez-Pastor ◽  
D.M. Barry ◽  
P. Bartels
Keyword(s):  
South Africa ◽  
Breeding Season ◽  
Plasma Membranes ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Assisted Reproduction Technology ◽  
Epididymal Spermatozoa ◽  
Connochaetes Taurinus ◽  
Free Ranging ◽  
Aepyceros 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<0.05) MS-IPM: 0.75; IPM-MIT: 0.83, and for blue wildebeest (n=6, P<0.05), MS-IPM: 0.84; IPM-MIT: 0.81, and for pooled data (n=14, P<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.)

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

2004 ◽  
Vol 16 (2) ◽  
pp. 227
Author(s):  
F. Olivier ◽  
T. Spies ◽  
F. Martinez-Pastor ◽  
D.M. Barry ◽  
P. Bartels
Keyword(s):  
Plasma Membrane ◽  
Assisted Reproduction ◽  
Sperm Quality ◽  
Osmotic Shock ◽  
Membrane Integrity ◽  
Molecular Probes ◽  
Assisted Reproduction Techniques ◽  
Plasma Membrane Integrity ◽  
Epididymal Spermatozoa ◽  
Connochaetes Taurinus

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<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)

217 QUALITY CHANGES IN POST-THAW SPRINGBOK (ANTIDORCAS MARSUPIALIS) EPIDIDYMAL SPERM MAINTAINED IN FERTILIZATION MEDIUM

2006 ◽  
Vol 18 (2) ◽  
pp. 216
Author(s):  
F. P. Chatiza ◽  
G. M. Pieterse ◽  
P. Bartels
Keyword(s):  
South Africa ◽  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Water Bath ◽  
Quality Changes ◽  
Epididymal Sperm ◽  
Plasma Membrane Integrity ◽  
Free Ranging ◽  
Over Time

The availability of gametes from the cropping of excess wildlife species provides the opportunity for the advancement of knowledge into assisted reproductive technology for possible future conservation measures. Little is known about the longevity of springbok (Antidorcas marsupialis) spermatozoa maintained in fertilization medium. The aim of this project was to determine the quality changes of post-thawed springbok spermatozoa incubated in fertilization medium by measuring plasma membrane integrity over time. Testes (n = 12) were obtained from two geographically distinct free-ranging springbok populations in South Africa. Spermatozoa were flushed from the cauda epididymides within three hours of the animals' death. Samples from an individual male were pooled, diluted to 400 × 106 sperm/mL with Biladyl (Minitüb, Tiefenbach, Germany) fraction A (no glycerol) and equilibrated in a water bath for 6 h at 4°C. An equal volume of Biladyl fraction B (containing 12% glycerol) was added to the sample to make a final concentration of 200 × 106 sperm/mL. Samples were loaded into 0.25-mL straws and frozen in liquid nitrogen vapor (5 cm above the liquid nitrogen level) for 20 min after which they were plunged into liquid nitrogen. Straws from each sample were thawed for 20 s at 36°C in a water bath. Thawed spermatozoa (100 μL) was added to 1 mL IVF-TALP medium containing heparin and PHE (Vajta et al. 1996 Theriogenology 45, 683–689) in 2-mL Nunc tubes (AEC, Amersham, South Africa) and incubated at 38.7°C, in humidified 5% CO2 balance air for 30 h. Aliquots were extracted from the incubating spermatozoa to determine plasma membrane integrity at 6-h intervals. Propidium iodide (Sigma, South Africa) at 50 ng/mL (10 min at RT) was used to evaluate membrane integrity under fluorescence microscopy at ×400, with a 450-nm excitation filter, a 510-nm dicroic beam splitter, and a 520-nm barrier filter. Cells with damaged plasma membrane have nuclei that fluorescence red. Eosin/nigrosin was also used to evaluate membrane integrity under ×400 bright-field microscopy. Cells with damaged plasma membrane stain purple-red, whereas the balance of cells remain translucent. The average post-thaw motility of spermatozoa in populations A and B was 69% (n = 6) and 68% (n = 6), respectively. Plasma membrane integrity of post-thawed springbok spermatozoa deceased steadily in IVF-TALP medium over the 30-h period (Table 1). Cryopreserved epididymal sperm derived from free-ranging springbok populations survive in IVF-TALP media and may be useful in future conservation activities where an isolated gene pool requires genetic supplementation through one or more assisted reproduction techniques such as IVF or AI. Further research is required to confirm and extend these findings. Table 1. Percentage plasma membrane integrity of post-thawed springbok sperm over time

scholarly journals Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls

Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1135
Author(s):  
Jesús L. Yániz ◽  
Inmaculada Palacín ◽  
Miguel A. Silvestre ◽  
Carlos Olegario Hidalgo ◽  
Carolina Tamargo ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Low Fertility ◽  
Head Width ◽  
Low Field ◽  
Frozen Semen ◽  
Acrosome Integrity ◽  
Sperm Acrosome ◽  
Acrosomal Integrity

The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested; 10 bulls had high field fertility and 10 bulls had low field fertility. Analyses of sperm motility, membrane integrity, and membrane–acrosome integrity with the ISAS3Fun method were performed. Sperm morphometry of the fluorescence sperm subpopulations obtained with the ISAS3Fun method was also analysed. Significant differences between high- and low-fertility groups were only found with the ISAS3Fun technique, specifically in sperm acrosome integrity, the proportion of spermatozoa with an intact acrosome and damaged membrane, and in sperm head width of spermatozoa with intact structures. Discriminant analyses allowed us to correctly classify 90% of sperm samples in their fertility group using sperm quality parameters. Given that only the results obtained with the ISAS3Fun technique were related to bull fertility, we performed a second experiment aimed to validate the efficacy of this technique to detect the acrosomal integrity of bull spermatozoa, comparing them with the conventional FITC-PNA/propidium iodide (PNA/PI) combination under capacitating conditions. The results indicated that the ISAS3Fun combination provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa, while the PNA/PI combination underestimated the extension of acrosomal damage due to false negatives. It was concluded that the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than more conventional in vitro sperm quality tests.

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

2004 ◽  
Vol 16 (2) ◽  
pp. 225 ◽  
Author(s):  
A.F. Malo ◽  
F. Martinez-Pastor ◽  
F. Olivier ◽  
T. Spies ◽  
E.R.S. Roldan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Osmotic Shock ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Storage Period ◽  
Egg Yolk ◽  
Plasma Membrane Integrity ◽  
Sperm Plasma Membrane ◽  
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.

scholarly journals Macro- and microelements in serum and seminal plasma as biomarkers for bull sperm cryotolerance

2021 ◽  
Vol 63 (1) ◽  
Author(s):  
Maja Zakošek Pipan ◽  
Petra Zrimšek ◽  
Breda Jakovac Strajn ◽  
Katarina Pavšič Vrtač ◽  
Tanja Knific ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Characteristics ◽  
Freezing And Thawing ◽  
Additional Tool ◽  
Bull Semen ◽  
Bull Sperm ◽  
Fresh Semen

ABSTRACT Background Wide variation in fertility rates is observed when using frozen bull semen, even when the bulls have met quality standards for semen production. Therefore, a simple and reliable test to assess the freezing potential of bull semen based on the analysis of fresh semen or blood would be of great value. Attention is now turning to assessment of seminal plasma components such as proteins and elements. In the present study, the concentrations of macro- and microelements in fresh bull semen plasma and in serum and their correlation with quality characteristics of fresh semen and with semen quality after freezing and thawing were determined. Ejaculates were collected from 30 mature bulls, and semen volume, concentration, sperm motility, morphology, tail membrane integrity, plasma membrane permeability and DNA fragmentation were determined on the day of collection and after freezing and thawing. The concentrations of macroelements (Na, Mg, K and Ca) and microelements (Cu, Fe, Zn and Se) were determined in the seminal plasma and serum. The semen samples were classified into satisfactory and unsatisfactory groups according to the fresh semen quality. Results Zinc and Se levels measured in serum were associated with almost all fresh and frozen-thawed semen quality characteristics, while Fe levels were associated only with acrosomal defects in fresh semen. Zinc and Fe levels in fresh seminal plasma were associated with various quality characteristics of fresh and frozen-thawed semen, while Se level in fresh seminal plasma was not associated with any of the semen quality characteristics. Conclusions Microelements were shown to be useful as biomarkers involved in the analysis of bull sperm quality and could be used as an additional tool to predict bull semen quality after freezing and thawing. Our results confirm that the analysis of Zn and Se levels in serum and Zn, Cu and Fe levels in fresh seminal plasma can provide information to discriminate between bull semen samples with spermatozoa with high or low cryotolerance.

scholarly journals Ovarian cycle activity varies with respect to age and social status in free-ranging elephants in Addo Elephant National Park, South Africa

2013 ◽  
Vol 1 (1) ◽  
pp. cot025-cot025 ◽  
Author(s):  
E. W. Freeman ◽  
J. M. Meyer ◽  
S. B. Putman ◽  
B. A. Schulte ◽  
J. L. Brown
Keyword(s):  
South Africa ◽  
Social Status ◽  
National Park ◽  
Ovarian Cycle ◽  
Free Ranging

scholarly journals Foraging range and habitat use by Cape Vulture Gyps coprotheres from the Msikaba colony, Eastern Cape province, South Africa

Koedoe ◽  
2015 ◽  
Vol 57 (1) ◽  
Author(s):  
Morgan B. Pfeiffer ◽  
Jan A. Venter ◽  
Colleen T. Downs
Keyword(s):  
South Africa ◽  
Protected Areas ◽  
Breeding Season ◽  
Movement Ecology ◽  
Kernel Density ◽  
Eastern Cape Province ◽  
Eastern Cape ◽  
Convex Polygons ◽  
Density Estimates ◽  
Kernel Density Estimates

Despite the extent of subsistence farmland in Africa, little is known about endangered species that persist within them. The Cape Vulture (Gyps coprotheres) is regionally endangered in southern Africa and at least 20% of the population breeds in the subsistence farmland area previously known as the Transkei in the Eastern Cape province of South Africa. To understand their movement ecology, adult Cape Vultures (n = 9) were captured and fitted with global positioning system/global system for mobile transmitters. Minimum convex polygons (MCPs),and 99% and 50% kernel density estimates (KDEs) were calculated for the breeding and non breeding seasons of the Cape Vulture. Land use maps were constructed for each 99% KDE and vulture locations were overlaid. During the non-breeding season, ranges were slightly larger(mean [± SE] MCP = 16 887 km2 ± 366 km2) than the breeding season (MCP = 14 707 km2 ± 2155 km2). Breeding and non-breeding season MCPs overlapped by a total of 92%. Kernel density estimates showed seasonal variability. During the breeding season, Cape Vultures used subsistence farmland, natural woodland and protected areas more than expected. In the non-breeding season, vultures used natural woodland and subsistence farmland more than expected, and protected areas less than expected. In both seasons, human-altered landscapes were used less, except for subsistence farmland.Conservation implications: These results highlight the importance of subsistence farm land to the survival of the Cape Vulture. Efforts should be made to minimise potential threats to vultures in the core areas outlined, through outreach programmes and mitigation measures.The conservation buffer of 40 km around Cape Vulture breeding colonies should be increased to 50 km.

The Influence of Cigarette Smoking on Sperm Quality and Sperm Membrane Integrity

2016 ◽  
Vol 12 (1) ◽  
pp. 58-65 ◽  
Author(s):  
Nyaz Shelko ◽  
Mohammed F. Hamad ◽  
Mathias Montenarh ◽  
Mohamam E. Hammadeh
Keyword(s):  
Cigarette Smoking ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Sperm Membrane

scholarly journals Visitation patterns of principal species of the insect-complex at carcasses in the Kruger National Park

Koedoe ◽  
1981 ◽  
Vol 24 (1) ◽  
Author(s):  
L.E.O Braack
Keyword(s):  
South Africa ◽  
National Park ◽  
Kruger National Park ◽  
Physical Attributes ◽  
Principal Species ◽  
Aepyceros Melampus

Two full-grown impala rams Aepyceros melampus were shot on 1978.01.07 in the Pafuri area of the Kruger National Park, Republic of South Africa. The carcasses were placed in enclosures 2,7 km apart and used to monitor the visitation patterns of insects. Collections of insects were made at four-hourly intervals for the first six days after placement of the carcasses, and thereafter every six hours up to the eleventh and final day. A figure is given to describe changes in the physical attributes of the carcasses through time. Twelve figures depict the patterns of arrival of insects at the carrion habitat. Species from the following families are represented: Cleridae, Dermestidae, Histeridae, Scarabaeidae, Silphidae, Staphylinidae, Trogidae (Coleoptera); Calliphoridae, Muscidae, Piophilidae, Sepsidae (Diptera); Diapriidae and Formicidae (Hymenoptera). The results indicate that species have distinctive periods of abundance and presents an overall picture of insect succession at carrion.

Sign in / Sign up

Export Citation Format

Share Document