71 EFFECT OF CHOLESTEROL-LOADED CYCLODEXTRINS ON PRE- AND POST-THAW FUNCTION OF FELID SPERM

2015 ◽  
Vol 27 (1) ◽  
pp. 128 ◽  
Author(s):  
I. A. Plourde ◽  
H. L. Bateman ◽  
W. F. Swanson
Keyword(s):  
Sperm Motility ◽  
Calcium Ionophore ◽  
Fluorescein Isothiocyanate ◽  
Cholesterol Content ◽  
Domestic Cat ◽  
Sperm Cryopreservation ◽  
Soy Lecithin ◽  
Acinonyx Jubatus ◽  
Cholesterol Levels

Propagation of genetically diverse felid populations would benefit from more effective assisted reproduction strategies, including enhanced methods for sperm cryopreservation. In felids, sperm cryopreservation has been improved by substituting soy-lecithin for egg yolk in cryomedium (Vick et al. 2012 Theriogenology 78, 2120–2128). In other species, such as elephants (Kiso et al. 2012 Reprod., Fert. Dev. 24, 1134–1142) and cattle (Purdy et al. 2004 Cryobiology 48, 36–45), the addition of cholesterol-loaded cyclodextrins (CLC) to sperm before freezing has been shown to produce superior cryopreservation results. In this study, our objectives were to (1) assess cholesterol content of cat sperm membranes and capacitation status following incubation with CLC; (2) evaluate post-thaw sperm motility, acrosome status, and fertility in vitro following CLC treatment and freezing in a soy-based cryomedium; and (3) conduct a preliminary assessment of cholesterol content in nondomestic cat sperm. Freshly collected domestic cat sperm (n = 2 males, 3–4 ejaculates/male) were incubated with CLC (0, 1.5, or 3.0 mg mL–1), and cholesterol levels were measured using an Amplex Red Cholesterol Assay. Sperm aliquots from each CLC concentration were treated with calcium ionophore (2 μM, 30 min) during in vitro incubation and stained with fluorescein isothiocyanate/PNA to evaluate induced acrosomal loss. To assess post-thaw parameters, cat sperm treated with CLC were frozen in straws using soy-lecithin cryomedium, thawed, and cultured in vitro over time. To evaluate fertility, oocytes were collected laparoscopically from gonadotropin-treated domestic cats (n = 7 females, 147 oocytes total) and inseminated with low numbers of thawed-frozen sperm pretreated with 0 or 1.5 mg mL–1 CLC. Data were analysed using ANOVA and mean differences assessed with Fisher l.s.d. or chi-squared analysis. Sperm cholesterol levels were increased (P < 0.05) after exposure to both 1.5 and 3.0 mg mL–1 CLC. Prefreeze motility was decreased (P < 0.05) and capacitation was delayed at 3.0 mg mL–1 CLC relative to treatment with 0 or 1.5 mg mL–1 CLC. Both post-thaw motility and percentage of acrosome intact sperm were reduced (P < 0.05) with the highest CLC concentration, but results were similar (P > 0.05) for 0 and 1.5 mg mL–1 CLC. Fertilization percentages did not differ (P > 0.05) between treatment groups (0 CLC, 33.3%, 25/75; 1.5 mg mL–1 CLC, 26.4%, 19/72). Preliminary results from a single cheetah (Acinonyx jubatus) and single fishing cat (Prionailurus viverrinus) suggest that sperm membrane cholesterol may be lower compared to the domestic cat. Cholesterol content appeared to increase in both species after exposure to 1.5 mg mL–1 CLC. In summary, our findings suggest CLC treatment increased cholesterol content of felid sperm membranes. The higher CLC concentration was detrimental to sperm motility, capacitation, and post-thaw sperm traits. The lower CLC concentration did not improve post-thaw sperm function in domestic cats.Research supported by the Procter & Gamble Wildlife Conservation Scholarship Program.

scholarly journals Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda

Reproduction ◽  
2004 ◽  
Vol 127 (5) ◽  
pp. 547-556 ◽  
Author(s):  
R E Spindler ◽  
Y Huang ◽  
J G Howard ◽  
P Wang ◽  
H Zhang ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Giant Panda ◽  
Calcium Ionophore ◽  
Sperm Cryopreservation ◽  
Management Tools ◽  
Giant Pandas ◽  
Before And After ◽  
Cryopreservation Protocol ◽  
Acrosomal Integrity

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze–thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (− 40 and − 100 °C/min) cryopreservation by incubation in HEPES-buffered Ham’s F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean±s.e.m. sperm motility post-thaw (56.1 ± 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 ± 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 ± 1.7% versus cryopreserved–thawed, 81.7 ± 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 ± 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 ± 1.1%). Frozen–thawed sperm preincubated without accelerators underwent capacitation (49.6 ± 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 ± 1.4%) and without accelerators (9 h: 41.2 ± 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

41 EFFICIENT STRATEGY FOR INTERSPECIFIC CLONING IN FELIDS

2014 ◽  
Vol 26 (1) ◽  
pp. 134
Author(s):  
L. N. Moro ◽  
M. I. Hiriart ◽  
J. Jarazo ◽  
C. Buemo ◽  
A. Sestelo ◽  
...  
Keyword(s):  
Species Conservation ◽  
Total Cell ◽  
Domestic Cat ◽  
Control Group ◽  
Felis Silvestris ◽  
Blastocyst Formation ◽  
Acinonyx Jubatus ◽  
Cell Numbers ◽  
Number Of Cells

Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) comes as a strategy to contribute to these species conservation. The aim of this study was to evaluate the effect of embryo aggregation in cheetah (Ch, Acinonyx jubatus), bengal (Ben, a hybrid between Felis silvestris and Prionailurus bengalensis), and domestic cat (DC, Felis silvestris) embryos generated by cloning. DC oocytes were in vitro matured and zona-free SCNT (with DC fibroblasts) or iSCNT (with Ch or Ben fibroblasts) was performed. The reconstructed embryos were activated with 5 μM ionomycin and 1.9 mM 6-DMAP, and cultured in SOF using microwells. Cloned embryos were cultured individually or as 2-embryo aggregates. The experimental groups were Ch1X, Ch2X, Ben1X, Ben2X, and the control groups were DC1X and DC2X. Embryo development was compared by Fisher's exact test (P ≤ 0.05). Embryo aggregation improved cleavage (Day 2) and blastocyst (Day 7) rates per well in all the groups (87.2% v. 96.7%, 83.8% v. 93.3% and 87.6% v. 98.2% for cleavage; and 13.7% v. 28.6%, 33.3% v. 43.8% and 27.4% v. 47.7% for blastocyst, for Ch1X (n = 102), Ch2X (n = 91), Ben1X (n = 154), Ben2X (n = 105), DC1X (n = 113), and DC2X (n = 109), respectively. Moreover, the Ch2X blastocyst rate was statistically similar as the control group DC1X. The mean total cell numbers of the blastocysts obtained were 264 ± 211 and 400.8 ± 97 for Ch1X and Ch2X, 278 ± 62 and 517 ± 104 for Ben1X and Ben2X, 385 ± 127 and 625 ± 183 for DC1X and DC2X, respectively. Although no statistical differences were obtained between the 1X and 2X groups, the 2X groups nearly doubled the average number of cells compared with the 1X groups. Blastocysts were also classified as grade 1 (expanded blastocysts with a well-defined ICM), grade 2 (expanded blastocysts without a well-defined ICM), and grade 3 (not expanded blastocysts). This classification showed an increase in grade 1 DC2X blastocyst compared with DC1X blastocysts (36.7% v. 16.1%), but no differences were observed in the other species. Expression of OCT-4 was assessed by inmunocytochemistry. The cheetah blastocysts markedly over-expressed this protein: the percentage of cells that expressed OCT-4 in Ch1X, Ch2X, Ben1X, Ben2X, DC1X, and DC2X was 88.2, 80.2, 46.3, 45.4, 51, and 47.4%, respectively, with statistical differences among all the groups except Ben1X and Ben2X. The proportion of OCT-4 expressing cells over total cell numbers was analysed by the difference of proportions test (P ≤ 0.05). In conclusion, iSCNT resulted in high rates of blastocyst formation, especially when embryo aggregation was applied. This strategy has not been previously evaluated in felids or iSCNT procedures, and has been demonstrated to improve blastocyst formation, the number of cells in the 3 groups, and the blastocyst quality in the DC. Other pluripotent genes besides OCT-4 should be studied to determine whether the overexpression of this gene in cheetah embryos is the consequence of an inefficient nuclear reprogramming that prevents a correct regulation. Finally, the iSCNT and embryo aggregation could contribute to species conservation in felids.

212 INTERSPECIFIC CLONING AND EMBRYO AGGREGATION INFLUENCE THE EXPRESSION OF oct4, nanog, sox2, AND cdx2 IN CHEETAH AND DOMESTIC CAT BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 196
Author(s):  
L. N. Moro ◽  
D. Veraguas ◽  
L. Rodriguez-Alvarez ◽  
M. I. Hiriart ◽  
C. Buemo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Internal Control ◽  
Gene Expression Analysis ◽  
Early Embryo ◽  
Domestic Cat ◽  
Nuclear Reprogramming ◽  
Acinonyx Jubatus ◽  
Standard Curve

The cheetah (Ch, Acinonyx jubatus) is a species considered globally endangered and cloning is one of the assisted reproductive techniques that can help to preserve it and to study early embryo development. However, the production of cloned felid embryos remains inefficient, probably because of the difficulty to control the process of nuclear reprogramming and obtain adequate gene expression. Embryo aggregation has been demonstrated to improve the cloning efficiency in several species and to normalise cdx2 in the mouse by lowering its expression (Balbach et al. 2010), but it has not been evaluated in felids before. To better understand the effect of interspecific somatic-cell nuclear transfer (iSCNT) and embryo aggregation in nuclear reprogramming, we analysed the expression of oct4, sox2, nanog, and cdx2 in cheetah blastocysts generated by iSCNT, domestic cat blastocysts (Dc) generated by SCNT, and IVF blastocysts as control. To achieve this, domestic cat oocytes were in vitro matured and zona-free SCNT or iSCNT was performed, as previously described (Moro et al. 2014, Reprod. Fertil. Dev.). Zona-free reconstructed embryos were then cultured individually (1X) or two embryo were cultured together (2X) in microwells, in synthetic oviductal fluid (SOF) medium. The experimental groups were Dc1X, Dc2X, Ch1X, Ch2X, and IVF. After 8 days of in vitro culture the blastocysts obtained were stored in RNA-later at –20°C. For gene expression analysis, blastocysts were pooled as follows: Dc1X, 4 replicates of 3 blastocysts each; Dc2X, 4 replicates of 3 blastocysts each; Ch1X, 2 replicates of 2 blastocysts and 1 replicate of 1 blastocyst; Ch2X, 4 replicates of 3 blastocysts each; IVF 3 replicates of 3 blastocysts each. Embryos were treated with a Cells-to-cDNA TM II kit (Life Technologies, Carlsbad, CA, USA) lyses buffer and treated with DNase I (0.04 U μL–1) for genomic DNA digestion. Gene expression analysis was performed by real-time qPCR using the standard curve method. In all qPCRs, GAPDH was used as an internal control. The statistical analysis was performed using a non-parametric Kruskal–Wallis test (P < 0.05). We observed that Dc1X blastocysts overexpressed the 4 genes evaluated respect to the IVF control. However, the gene expression of the aggregated group (Dc2X) was lower for all the genes, achieving the same levels of nanog and sox2 as the IVF blastocysts. The expression of oct4 and cdx2 were also closer to the expression levels of the control in the Dc2X group than in the Dc1X group. With respect to interspecific embryos, the amount of oct4 and cdx2 was also significantly reduced in the Ch2X blastocysts respect to Ch1X blastocysts. Both cheetah groups showed significantly lower expression of oct4, cdx2, and nanog than the IVF control. In conclusion, transcription of pluripotent and early differentiation factors in cheetah embryos was not as efficient as in the domestic cat embryos, probably caused by interspecific transfer. Our study demonstrated for the first time that defects in gene expression of domestic cat embryos can be corrected by embryo aggregation, providing a simple strategy to improve felid cloning.

97 IMPROVED CRYOPRESERVATION OF DOMESTIC CAT SPERMATOZOA IN A SOY LECITHIN-BASED EXTENDER

2011 ◽  
Vol 23 (1) ◽  
pp. 153 ◽  
Author(s):  
M. M. Vick ◽  
H. L. Bateman ◽  
W. F. Swanson
Keyword(s):  
Sperm Motility ◽  
Room Temperature ◽  
Egg Yolk ◽  
Tris Buffer ◽  
Domestic Cat ◽  
Dna Integrity ◽  
Freezing And Thawing ◽  
Soy Lecithin ◽  
Acridine Orange Staining ◽  
Acrosome Integrity

Development of a chemically defined, plant-based cryopreservation media would reduce extender variability and the potential for transmission of zoonotic pathogens compared with traditional egg-yolk-based extenders. The objective of this study was to compare effects of egg yolk- and soy lecithin-based cryopreservation media and the temperature of glycerol addition on sperm parameters following freezing and thawing of domestic cat spermatozoa. Fresh semen was collected by manual stimulation on 3 separate occasions from 4 adult male cats. Each ejaculate was washed to remove seminal plasma, divided into 4 equal aliquots, and extended at room temperature in one of the following treatments: 1) TEST-egg yolk (Irvine Scientific Inc., Santa Ana, CA, USA) medium with 4% glycerol (EYG); 2) TEST-egg yolk, with 4% glycerol added after cooling to 5°C (EY); 3) TES-Tris buffer with soy-lecithin (1%) and 4% glycerol (SLG); and 4) TES-Tris buffer with 1% soy-lecithin, and 4% glycerol added after cooling to 5°C (SL). Sperm progressive motility (%) and rate of progressive movement (scale of 0–5) were evaluated at 0, 1, 3, 6, and 24 h post-thaw. Sperm capacitation (chlortetracycline staining), acrosome integrity (FITC-PNA staining), and DNA integrity (acridine orange staining) were assessed at 15 min post-thaw. Data were exponentially transformed to achieve normal distribution and then subjected to GLM analysis to determine effects of media and temperature of glycerol addition on sperm traits. At 0 and 1 h post-thaw, acrosome integrity, DNA integrity and % sperm motility did not differ (P > 0.05) among treatments. However, % sperm motility was greater in the soy-based media compared to egg yolk-based media at 3, 6, and 24 h post-thaw (Table 1; P < 0.05). A higher percentage of uncapacitated spermatozoa were present in soy-based compared to egg-yolk based cryopreservation media (63.9 ± 9.3 v. 51.2 ± 11.5, respectively; P < 0.05), regardless of temperature of glycerol addition. Finally, addition of glycerol at 5°C resulted in higher % sperm motility compared to room temperature at 6 and 24 h post-thaw in both medium types (Table 1; P < 0.05). Our results suggest that use of a chemically defined, soy-based medium improves long-term motility and capacitation status of frozen–thawed domestic cat spermatozoa compared with cryopreservation in a traditional egg yolk-based extender. Table 1.Motile spermatazoa and motility score at 3, 6, and 24 h

102 Sperm cryopreservation with a soy lecithin-based medium in black-footed cats (Felis nigripes) and sand cats (Felis margarita)

2019 ◽  
Vol 31 (1) ◽  
pp. 177
Author(s):  
L. M. Vansandt ◽  
A. Moresco ◽  
R. González ◽  
A. Miller ◽  
J. Newsom ◽  
...  
Keyword(s):  
Egg Yolk ◽  
Animal Protein ◽  
Domestic Cat ◽  
Significant Advance ◽  
Sperm Cryopreservation ◽  
Soy Lecithin ◽  
Sand Cat ◽  
Embryo Cleavage ◽  
Blastomere Number ◽  
Time Point

Felid semen has historically been frozen using an egg yolk-based cryopreservation medium (TEY). However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. Our recent research has focused on developing an animal protein-free medium containing soy lecithin (SOY). Our studies revealed that SOY was superior to TEY for freezing domestic cat sperm and provided similar results for freezing ocelot, Pallas’ cat, and fishing cat sperm. The objective of this study was to compare SOY to the standard TEY for sperm cryopreservation in 2 wild cat species: the black-footed cat and sand cat. Semen was collected from adult male cats (n=6/species) via electroejaculation, split into 2 aliquots, centrifuged, resuspended in either SOY or TEY, slow-cooled, and frozen in straws over nitrogen vapor. Sperm motility [percent progressively motile (PPM); rate of progressive motility on 0-5 scale (RPM)] was evaluated at 0, 1, 3, 6, and 24h post-thaw and acrosome status (AC) was assessed at 0 and 6h post-thaw. Heterologous IVF was performed using oocytes collected laparoscopically from gonadotropin-treated domestic cats. At 48h post-insemination, Hoechst33342 staining was used to determine oocyte stage, number of blastomeres, and number of accessory sperm (AS) bound to the zona pellucida of embryos and mature oocytes. Percent progressively motile, RPM, and AC were analysed with repeated-measures ANOVA; embryo cleavage, blastomere number, and AS number were analysed with one-way ANOVA. All data are reported as least squares means±average standard error. In the black-footed cat, PPM, RPM, and AC of SOY-treated sperm (32.5±4.0% motile, 2.8±0.2 RPM, 41.8±4.1% intact; 0h) did not differ from TEY-treated sperm (44.2±4.0% motile, 2.8±0.2 RPM, 46.8±4.1% intact; 0h) at any post-thaw time point (P &gt; 0.05). Similarly, in the sand cat, post-thaw PPM, RPM, and AC of SOY-treated sperm (36.7±5.2% motile, 2.6±0.2 progression, 53.3±5.8% intact; 0h) did not differ from TEY-treated sperm (45.8±5.2% motile, 2.8±0.2 RPM, 51.0±5.8% intact; 0h) at any time point (P &gt; 0.05). In black-footed cats, neither embryo cleavage (34.1±10.9% SOY; 58.5±10.9% TEY), blastomere number (7.8±0.8 SOY; 6.3±0.8 TEY), nor AS (3.5±0.8 SOY; 1.7±0.8 TEY) differed between treatments (P &gt; 0.05). Sand cat results were similar, with no difference between SOY and TEY for cleavage (44.7±10.8% SOY; 40.6±10.8% TEY) or blastomere number (7.4±2.0 SOY; 6.7±2.0 TEY) (P &gt; 0.05), but AS was higher in SOY-treated sperm (4.3±0.2 SOY; 3.5±0.2 TEY, P=0.0183). These data collectively demonstrate that our SOY medium was an effective substitute to TEY for sperm cryopreservation in the black-footed cat and sand cat. The replacement of an egg yolk-based cryomedium with a chemically defined, animal protein-free alternative represents a significant advance in quality control and biosecurity for felid semen banking and should augment the use of assisted reproduction for population management of imperiled cats. Funded by the Institute of Museum and Library Services.

Spermatozoa from mice deficient in Niemann-Pick disease type C2 (NPC2) protein have defective cholesterol content and reduced in vitro fertilising ability

2014 ◽  
Vol 26 (4) ◽  
pp. 609 ◽  
Author(s):  
Dolores Busso ◽  
María José Oñate-Alvarado ◽  
Elisa Balboa ◽  
Juan Castro ◽  
Carlos Lizama ◽  
...  
Keyword(s):  
Cholesterol Content ◽  
Disease Type ◽  
Wild Type ◽  
Cholesterol Levels ◽  
Niemann Pick Disease ◽  
Epididymal Maturation ◽  
Sperm Membrane ◽  
Niemann Pick ◽  
Pick Disease

The cholesterol content of the sperm membrane is regulated during both maturation in the epididymis and capacitation in the female tract, two processes required for the spermatozoa to acquire their fertilising ability. Because Niemann-Pick disease, type C2 (NPC2) protein is one of the most abundant components of the epididymal fluid and contains a functional cholesterol-binding site that can transfer cholesterol between membranes, it has been suggested for years that NPC2 could be involved in the regulation of cholesterol levels in spermatozoa during epididymal maturation. In the present study, western blot and immunohistochemistry analyses demonstrated significant levels of NPC2 in the mouse epididymal epithelium. Epididymal spermatozoa obtained from NPC2–/– mice were morphologically normal and had normal motility parameters, but had a reduced cholesterol content compared with that of wild-type (WT) spermatozoa, as determined by both biochemical and by flow cytometry analyses. These results suggest that NPC2 could be involved in regulating cholesterol levels in spermatozoa during epididymal maturation. To understand the relevance of epididymal NPC2 for sperm function, the ability of spermatozoa to undergo events influenced by epididymal maturation, such as capacitation and fertilisation, were compared between WT and NPC2–/– mice. Capacitated NPC2–/– spermatozoa exhibited defective tyrosine phosphorylation patterns and a reduced ability to fertilise cumulus–oocyte complexes compared with WT spermatozoa, supporting the relevance of mouse epididymal NPC2 for male fertility.

scholarly journals Glycolytic Enzyme Activity Is Essential for Domestic Cat (Felis catus) and Cheetah (Acinonyx jubatus) Sperm Motility and Viability in a Sugar-Free Medium1

2011 ◽  
Vol 84 (6) ◽  
pp. 1198-1206 ◽  
Author(s):  
Kimberly A. Terrell ◽  
David E. Wildt ◽  
Nicola M. Anthony ◽  
Barry D. Bavister ◽  
S.P. Leibo ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Sperm Motility ◽  
Glycolytic Enzyme ◽  
Felis Catus ◽  
Domestic Cat ◽  
Acinonyx Jubatus

scholarly journals Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro

Reproduction ◽  
2005 ◽  
Vol 130 (6) ◽  
pp. 889-898 ◽  
Author(s):  
Hiroaki Funahashi
Keyword(s):  
Early Development ◽  
Penetration Rate ◽  
Calcium Ionophore ◽  
Fluorescein Isothiocyanate ◽  
Cortical Granules ◽  
Pixel Intensity ◽  
Vitro Fertilization ◽  
Sperm Penetration ◽  
Development In Vitro

This study was carried out to determine the effects of beta-mercaptoethanol (bME) during a transient co-culture of gametes for 10 min, and/or the following culture until 6–9 h after insemination, on sperm penetration of porcine in vitro maturation (IVM) oocytes and the early development in vitro. When fresh spermatozoa were cultured in various concentrations of bME for 2 h, bME neutralized the stimulatory effect of caffeine-benzoate on sperm capacitation and the spontaneous acrosome reaction at 50–250 μmol/l. When 50 μmol/l bME were added during a transient co-culture of gametes for 10 min, the sperm penetration rate was reduced 9 h after insemination (70.5–82.0% vs 90.5–94.0% in the absence of bME), but the incidence of monospermic penetration was not affected. When 50 μmol/l bME were supplemented during culture after a transient co-culture, the sperm penetration rate was not affected, but the incidence of monospermy oocytes was increased (43.9–45.8% vs 31.7–34.3% in the absence of bME). The presence of bME following a transient co-culture minimized a decrease of oocyte glutathione content at 6 h after insemination (7.9 pmol/oocyte before in vitro fertilization (IVF), 6.7 pmol/oocyte in the presence of bME vs 5.5 pmol/oocyte in the absence of bME). When the distribution of cortical granules was evaluated 1 h after activation with calcium ionophore, mean pixel intensity of fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) at the cortex region was lower in the oocytes activated and cultured in the presence of 50 μmol/l bME. Although the presence of 50 μmol/l bME during a transient co-culture for 10 min and the following culture did not increased blastocyst formation (29.6–37.7%), 50 μmol/l bME during the following culture significantly increased the mean cell numbers per blastocyst (73.3–76.4 vs 51.2 in the presence and absence of bME respectively). These results demonstrate that supplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.

Assessment of basic seminal characteristics, sperm cryopreservation and heterologous in vitro fertilisation in the fishing cat (Prionailurus viverrinus)

2006 ◽  
Vol 18 (3) ◽  
pp. 373 ◽  
Author(s):  
Khongsak Thiangtum ◽  
William F. Swanson ◽  
JoGayle Howard ◽  
Wanchai Tunwattana ◽  
Dakara Tongthainan ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Sperm Morphology ◽  
Domestic Cat ◽  
Ex Situ ◽  
In Vitro Fertilisation ◽  
Genetic Management ◽  
Slow Cooling ◽  
Breeding Programmes ◽  
Acrosomal Integrity

Conservation of the fishing cat, a threatened south-east Asian felid, could benefit from effective ex situ genetic management and breeding programmes, including the use of assisted reproduction. The aims of the present study were to: (1) characterise basal seminal traits of fishing cats in Thailand zoos; and (2) investigate the effect of cryopreservation on sperm motility, acrosomal integrity and in vitro function. Seminal traits were evaluated in electroejaculates collected from eight males. Spermatozoa were diluted in n-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid Tris (TEST)-yolk buffer (TYB) without glycerol, then diluted further with TYB with glycerol (4% final concentration) at either 25°C or after slow cooling to 5°C and frozen in straws over liquid nitrogen vapour. After thawing, sperm function was assessed by insemination of viable domestic cat oocytes. Fishing cat ejaculates averaged (± s.e.m.) 43.6 ± 14.2 × 106 motile spermatozoa with 33.5 ± 6.8% normal sperm morphology. Semen processing had a negligible effect (P > 0.05) on sperm motility and acrosomal integrity, but values were reduced (P < 0.05) after thawing. All thawed samples fertilised domestic cat oocytes, with 62.1% (36/58) of mature oocytes cleaving. Glycerol addition at 5°C resulted in higher (P < 0.05) post-thaw motility and intact acrosomes than glycerol addition at 25°C. In conclusion, good-quality ejaculates can be obtained from Thai fishing cats and their spermatozoa exhibit adequate function after cryopreservation for in vitro fertilisation procedures.

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