221 BASAL SEMINAL TRAITS AND IN VITRO FERTILIZATION IN THE SAND CAT (FELIS MARGARITA)

2006 ◽  
Vol 18 (2) ◽  
pp. 218 ◽  
Author(s):  
J. Herrick ◽  
K. Leiske ◽  
G. Magarey ◽  
W. Swanson
Keyword(s):  
Embryonic Development ◽  
Sperm Motility ◽  
Captive Breeding ◽  
Fresh Medium ◽  
Domestic Cat ◽  
Motile Sperm ◽  
Genetic Management ◽  
Sand Cat ◽  
Acrosomal Integrity

The sand cat (Felis margarita) is one of five small-sized cat species given priority for conservation in North American zoos. An improved understanding of sand cat reproductive biology would benefit captive breeding and facilitate use of assisted reproduction for genetic management. In this study, our objectives were to: (1) characterize basal seminal traits, (2) assess ovarian responses to exogenous gonadotropins, and (3) compare Ham's (HF10) F-10 with 5% fetal bovine serum (FBS) and feline optimized culture medium (FOCM) with 0.4% BSA for supporting gamete function and embryonic development in vitro. Semen was collected by electroejaculation from seven males (n = 10 ejaculates), washed, and resuspended (10 � 106 motile sperm/mL) in HF10 or FOCM for culture (6% CO2 in air at 38.7�C). Sperm motility (% motile and rate of forward progress, 0-5 scale) was evaluated at 0, 1, 3, and 6 h of culture and used to calculate a sperm motility index (SMI; [% + (5 * rate)]/2). Acrosomal integrity was evaluated by staining (fast green FCF-rose bengal) at 0 and 6 h. For IVF, ovarian follicles were aspirated laparoscopically from female sand cats (n = 4) treated at random times of the estrous cycle with 150 IU eCG and 100 IU hCG (84 h post-eCG) prior to oocyte recovery (25 h post-hCG). Grade 1 oocytes were co-incubated with 2 � 105 motile sperm/mL in HF10 (n = 32) or FOCM (n = 33) for 20 h before transfer to fresh medium. Resulting embryos were either cryopreserved (n = 42) at 30 h post-insemination (hpi) or cultured until Day 7 pi after being moved to fresh medium (FOCM with 5% FBS (n = 10) or HF10 (n = 6)) on Day 3 pi. Ejaculates contained (mean � SEM) 43.5 � 11.0 � 106 total spermatozoa, with 77.0 � 2.3% motility, 43.8 � 3.9% normal morphology, and 93.1 � 1.3% intact acrosomes. During 6 h of culture, SMI and % intact acrosomes declined (P < 0.05) slightly (SMI, 73.8-74.8 at 0 h and 68.5-68.8 at 6 h; % intact acrosomes, 87.1-87.6% at 0 h and 69.0-74.2% at 6 h), but similarly (P > 0.05) in both media. Females produced 24.3 � 5.6 follicles, with 19.3 � 5.1 total oocytes and 16.5 � 4.6 Grade 1 oocytes recovered per female. The proportions of oocytes cleaving at 20 and 30 hpi and the quality of the resulting embryos at 30 hpi were higher (P < 0.05) in FOCM (20 hpi, 76.5 � 8.7%; 30 hpi, 92.9 � 7.1%; 89.2 � 7.9% Grade 1) than in HF10 (20 hpi, 29.8 � 11.7%; 30 hpi, 55.9 � 20.6%; 62.9 � 7.2% Grade 1). Two blastocysts developed in FOCM (69.0 � 19.0 cells), but the final cell numbers of all cultured embryos were not different (P > 0.05) between FOCM (26.9 � 8.0 cells) and HF10 (19.3 � 6.4 cells). Compared to other small felid species, sand cats exhibited excellent seminal traits, gonadotropin-induced ovarian responses, and fertilization success in vitro. Although sperm motility and acrosomal integrity were similar in FOCM and HF10, the medium developed specifically for domestic cat embryos (FOCM) better supported IVF and early embryonic development. These results indicate that IVF with fresh spermatozoa could be a valuable tool for genetic management of captive sand cat populations. This work was supported by MAF D04ZO-72.

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.

59 EFFECT OF PELLET VOLUME AND THAWING TEMPERATURE ON VITRIFICATION EFFICACY WITH DOMESTIC CAT SEMEN COLLECTED VIA URETHRAL CATHETERIZATION

2017 ◽  
Vol 29 (1) ◽  
pp. 137
Author(s):  
A. Moresco ◽  
H. L. Bateman ◽  
J. Newsom ◽  
W. F. Swanson
Keyword(s):  
Sperm Motility ◽  
Domestic Cat ◽  
Sperm Function ◽  
Sample Collection ◽  
Urethral Catheterization ◽  
Treatment Groups ◽  
Chi Squared ◽  
Embryo Cleavage ◽  
Acrosomal Integrity

Historically, semen banking in felids has required sample collection via electroejaculation followed by sperm freezing in straws over LN2 vapor. Recent modifications include urethral catheterization of males treated with α-2 agonists for semen recovery and vitrification of cat sperm by suspension in a sucrose-based cryomedium and direct pelleting into LN2. In combination, these latter methods greatly simplify semen cryopreservation in cats but protocols need to be optimized for applied usage. In the present study, our goal was to assess the effect of 2 variables—pellet volume and thawing temperature—on post-thaw sperm motility, acrosome status, and in vitro fertility. Semen was collected from 3 males (3 ejaculates/male) via urethral catheterization under dexmedetomidine-ketamine anaesthesia. Sperm were diluted in Feline Optimized Culture Medium (FOCM), centrifuged (8 min; 300 × g), and resuspended in a soy-lecithin-based vitrification medium (with 0.2 M sucrose). After a 5-min equilibration, sperm was vitrified in 2 volumes (20 or 30 µL) by direct pipetting into LN2. Sperm pellets were thawed in FOCM at 1 of 2 temperatures (37 or 55°C) and the 4 treatment groups (20 µL-37°C, 20–55, 30–37, 30–55) assessed for percentage of progressively motile and acrosome intact sperm. To assess sperm function, additional 30-µL pellets were thawed at 37 or 55°C, and recovered sperm were used to inseminate in vitro-matured domestic cat oocytes (n = 10–25/ejaculate). At 48 h post-insemination, oocytes and embryos were fixed (1% NBF). Hoechst fluorescent stain (#33342) was used to evaluate embryo cleavage and maturation status of unfertilized ova. Sperm motility and acrosomal integrity percentages were analysed by ANOVA, and oocyte cleavage proportions were analysed by chi-squared. Mean (± SEM) progressive sperm motility post-thaw did not differ (P > 0.05) among treatments (38 ± 8, 34 ± 7, 41 ± 7, 32 ± 7% for 20 µL-37°C; 20–55, 30–37, and 30–55, respectively). Similarly, acrosomal integrity did not differ (P > 0.05) among treatments (26 ± 4, 25 ± 4, 17 ± 3, 17 ± 2% for 20 µL-37°C, 20–55, 30–37, and 30–55, respectively). Oocyte cleavage proportions did not differ (P > 0.05) between thawing temperatures for total inseminated oocytes but, after correcting for oocyte maturation status, was higher (P < 0.01) for samples thawed at 55°C (60%, 67/112) compared with 37°C (39%, 52/133). In summary, although variations in pellet volume and thawing temperature had minimal effect on sperm motility or acrosome status immediately post-thaw, sperm function appeared to be enhanced when vitrified pellets were thawed at a higher temperature. In vitro fertility success (~60% embryo cleavage) is comparable to values reported by our laboratory with conventionally collected and frozen cat semen, suggesting these newer methods may be suitable for applied usage in felids. This study was funded by the Institute of Museums and Library Services.

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.

284 COMPARATIVE FERTILITY OF FRESHLY-COLLECTED VERSUS FROZEN - THAWED SPERMATOZOA FOR IN VITRO FERTILIZATION IN THE FISHING CAT (PRIONAILURUS VIVERRINUS)

2006 ◽  
Vol 18 (2) ◽  
pp. 249
Author(s):  
G. Magarey ◽  
J. Herrick ◽  
K. Thiangtum ◽  
W. Tunwattana ◽  
W. Swanson
Keyword(s):  
Sperm Motility ◽  
Culture Medium ◽  
Culture Media ◽  
Egg Yolk ◽  
Ovarian Follicles ◽  
Blastocyst Stage ◽  
Motile Sperm ◽  
Vitro Fertilization ◽  
International Transport

Wild populations of fishing cats (Prionailurus viverrinus) in Southeast Asia are in decline, primarily due to habitat loss. Because the fishing cat population in North American zoos is small (n = 69) and inbred (F = 0.17) with relatively low genetic variation (86%), infusion of new founder genes from Asia is a conservation priority. Importation of cryopreserved semen for use with IVF and ET may offer one alternative to the international transport of living animals. In this study, our objectives were to (1) compare motility longevity of fresh vs. frozen-thawed fishing cat spermatozoa in two culture media, (2) evaluate ovarian responses to exogenous gonadotropins, and (3) assess development of IVF embryos produced with fresh vs. frozen-thawed spermatozoa. Raw semen was collected via electroejaculation from male fishing cats (n = 4), divided into groups, and washed. Two sperm pellets were resuspended in either Ham's F10 medium (HF10; with 5% FBS) or our feline optimized culture medium (FOCM; with 0.4% BSA); another pellet was diluted in TEST egg yolk, cooled to 5�C over 3 h, glycerated (4%), and cryopreserved in straws over LN2 vapor. Frozen sperm samples were thawed, washed, and diluted in either HF10 or FOCM. Fresh and frozen-thawed sperm motility (percent motile, rate of forward progress) in each medium (10 � 106 motile sperm/mL) was assessed (at 0, 1, 3, and 6 h) in microdrops under oil during culture (38�C; 6% CO2 in air). Female fishing cats (n = 10) were treated with exogenous gonadotropins (150 IU eCG, 100 IU hCG, 85-h interval) and ovarian follicles were aspirated laparoscopically. Recovered oocytes were inseminated with fresh (2 � 105 motile sperm/mL) or frozen-thawed (5 � 105 motile sperm/mL) spermatozoa in FOCM microdrops; resulting embryos were either cryopreserved or cultured in FOCM (with 5% FBS added at 72 h post-insemination) for 7 days. Sperm motility over time did not differ (P > 0.05) between media for either fresh or frozen-thawed samples; however, across media, frozen-thawed sperm motility was lower (P < 0.05) and declined faster (P < 0.05) compared to fresh spermatozoa. Females produced an average (�SEM) of 9.8 � 2.9 mature ovarian follicles, allowing recovery of 7.3 � 2.6 high-quality oocytes per female. Oocyte cleavage percentage at 42 h p.i. was lower (P < 0.05) with frozen-thawed spermatozoa (38%, 11/29) compared to freshly collected spermatozoa (68%, 17/25). Overall, 35% (6/17) of cultured embryos developed to blastocysts with no difference (P > 0.05) between embryos produced with frozen-thawed (4/11) vs. fresh (2/6) spermatozoa. Although fishing cat sperm motility and fertility appear compromised after cryopreservation, our results demonstrate the ability of frozen-thawed spermatozoa to produce IVF embryos that are capable of developing to blastocyst stage in vitro. This work was supported by (NIH RR015388).

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.

116 DEVELOPMENT OF ASSISTED REPRODUCTION TECHNOLOGIES FOR THE ENDANGERED MISSISSIPPI GOPHER FROG (RANA SEVOSA) AND SPERM TRANSFER FOR IN VITRO FERTILIZATION

2012 ◽  
Vol 24 (1) ◽  
pp. 170 ◽  
Author(s):  
A. Kouba ◽  
E. Willis ◽  
C. Vance ◽  
S. Hasenstab ◽  
S. Reichling ◽  
...  
Keyword(s):  
Captive Breeding ◽  
Assisted Reproductive Technologies ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Critically Endangered ◽  
Leopard Frog ◽  
Genetic Management ◽  
Mississippi Gopher Frog

Species-specific differences in breeding strategies and physiology have limited the application of assisted reproductive technologies (ART) for critically endangered amphibians in captive assurance colonies. In 2006, the Memphis Zoo (MZ) initiated a program to develop ART for the critically endangered Mississippi gopher frog after natural breeding failed. Standard gamete collection and IVF developed by MZ for reproducing endangered toads such as the Wyoming or boreal toad were applied to the gopher frog with little success, especially hormonal therapy for sperm production. Using the leopard frog as a model species for Ranids, we tested the time and dose dependence of a luteinizing hormone releasing hormone analogue (LHRHa) and hCG on sperm quantity and quality. Initial findings from the leopard frog study were critical in designing the study on gopher frogs. Our objectives were to (1) compare 2 different hormones administered intraperitoneal (500 IU hCG vs 15 μg LHRHa) or their combination on spermiation in gopher frogs; (2) develop in vivo oocyte maturation and ovulation protocols using LHRHa (15 μg) and hCG (500 IU); and (3) transfer this technology to another institution as proof of principle. In gopher frogs, 100 and 83% of the males produced sperm in response to the LHRHa and the combination treatment, respectively, whereas only 16% responded to hCG alone. Sperm concentration peaked at 1 h post-administration for all treatments, with the LHRH/hCG cocktail treatment producing the highest concentration of sperm (mean = 4.6 × 106 ± 1.2 × 106 sperm mL–1, n = 6). No differences in motility were observed between treatments (P > 0.05). For females, a series of priming hormones of hCG and LHRHa were given several months before an ovulatory hormone regimen resulting in ovulation by 100% of the females (n = 6), whereas animals not primed failed to ovulate (n = 4). These 3 separate priming and IVF trials conducted between 2008 and 2010 resulted in each female laying ∼2000 eggs, with an average fertilization rate of 76% for inseminated eggs and hundreds of tadpoles produced. These IVF tadpoles represent the first captive reproduction of gopher frogs and highlight how ART can be applied to conservation and genetic management of threatened species. Subsequently, we tested our IVF protocols on gopher frogs at Omaha's Henry Doorly Zoo using fresh (collected on site) and chilled, shipped sperm from MZ. We collected 6169 eggs from 9 hormone-primed females with all animals ovulating. A portion of the total eggs ovulated were inseminated, resulting in 2401 fertilized eggs (38.9% of total eggs collected) across 18 different male–female pairings leading to viable tadpoles. In addition, sperm transferred overnight from the MZ produced 202/441 fertilized eggs (46%). The transfer of this technology and production of endangered amphibians using chilled, shipped sperm from live animals is a conservation milestone that can be applied to other captive breeding programs.

Embryo development capacity of oocytes fertilized by immature sperm and sperm treated with motility stimulants

1994 ◽  
Vol 6 (1) ◽  
pp. 113 ◽  
Author(s):  
O Lacham-Kaplan ◽  
AO Trounson
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Sperm Motility ◽  
Cumulus Cells ◽  
Progressive Motility ◽  
Development Capacity ◽  
Fertilizing Ability ◽  
Embryonic Arrest ◽  
Human And Mouse

The fertilizing ability of mouse spermatozoa develops during maturation and coincides with the acquisition of motility. The lack of progressive motility of spermatozoa from the testis and precaudal segments of the epididymis interferes with their ability to fertilize oocytes after insemination in vitro. The removal of cumulus cells for insemination in vitro and the use of subzonal injection of a single spermatozoon resulted in a higher number of oocytes fertilized by immature caput and corpus spermatozoa. High rates of embryonic arrest and retarded development were observed in oocytes fertilized by caput and corpus spermatozoa when compared with oocytes fertilized by cauda spermatozoa. However, when the oocytes were enclosed in their cumulus cells or microinjected with a single spermatozoon, these effects were reduced. A block in embryonic development was also observed after human and mouse oocytes were exposed to the sperm motility stimulants pentoxifylline (PTF) and 2-deoxyadenosine (DOA). These observations suggest that exposure of oocytes to PTF and DOA should be avoided.

151 EVALUATION OF REFERENCE GENE TRANSCRIPT STABILITY DURING EARLY PRE-IMPLANTATION DEVELOPMENT AND IN SPERMATOGONIAL CELLS IN THE CAT

2012 ◽  
Vol 24 (1) ◽  
pp. 187 ◽  
Author(s):  
M. N. Biancardi ◽  
C. E. Pope ◽  
R. H. Powell ◽  
J. Galiguis ◽  
C. Dumas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Reference Gene ◽  
18S Rrna ◽  
Domestic Cat ◽  
Amplification Efficiency ◽  
Gene Transcript ◽  
Sample Type ◽  
Selection Of

Differences in the stability of commonly used reference genes have been discovered between species and among different tissue types. These inconsistencies underscore the importance of validation and selection of an appropriate reference for normalization of gene expression to an endogenous control in RT-qPCR experiments. Three different reference transcripts (GAPDH, RPS19 and 18S rRNA) of 3 different functional categories were selected for evaluation during pre-implantation embryonic development and in spermatogonial cells in the domestic cat. Amplification efficiency was done by standard curve analysis with 5-fold template dilutions. For pre-implantation analysis, transcripts were assayed at 4 developmental stages: 2- to 6-cell (2–6C), 8- to 16-cell (8–16C), morula (M) and blastocyst (BL). Embryos were from pools of 4 to 10 in vitro matured and fertilized domestic cat embryos. For spermatogonia, isolates of 20 000 to 30 000 cells were assayed from single testis isolates following a collagenase and trypsin with DNase digestion and Percoll gradient separation. Total mRNA was isolated using the Cells-to-cDNA II Kit, with a minimum of 2 biological replicates for each sample type. The RNA quantitation was done by RiboGreen analysis and 47 ng of RNA was used for cDNA synthesis. Transcript abundance was detected in 2 technical replicates per sample by SYBR Green chemistry and data were analysed with NormFinder and one-way ANOVA. Each transcript showed varying levels of expression throughout development and among embryonic stages and in spermatogonial cells. The GADPH and 18S rRNA transcripts were expressed at higher levels in BL than in 2–6C and 8–16C, respectively. Contrarily, transcript levels of RSP19 were lower in BL compared with levels at 2–6C. Even though differences were observed among embryonic stages, analysis with NormFinder indicated that the most stable reference gene throughout early pre-implantation development was RPS19. When each stage was analysed separately, 18S rRNA was the most stable at 2–6C, whereas RPS19 was found to be the most stable at 8–16C, M and BL stages. In spermatogonial samples, GAPDH was the most stably expressed gene. Our results support the selection of an appropriate reference gene based on the needs of the experimental design. We conclude from these findings that when gene expression throughout the duration of early embryonic development is examined, RPS19 is the preferred selection. If gene expression is analysed within discrete time points of development only, it is appropriate to select 18S rRNA at the 2–6C stage and RPS19 for 8–16C, M and BL stage embryos. For spermatogonial samples, if comparing only different biological replicates, GAPDH should be selected for normalization of expression; however if the purpose of the experiment is to assay genes that are also expressed in BL, the most stably expressed reference gene is RPS19.

scholarly journals RFRP3 influences basal lamina degradation, cellular death, and progesterone secretion in cultured preantral ovarian follicles from the domestic cat

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7540 ◽  
Author(s):  
Kathryn Wilsterman ◽  
George E. Bentley ◽  
Pierre Comizzoli
Keyword(s):  
Basal Lamina ◽  
Captive Breeding ◽  
Direct Action ◽  
Ovarian Follicle ◽  
Ovarian Follicles ◽  
Domestic Cat ◽  
Follicle Development ◽  
Carnivore Species ◽  
Percentage Survival

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro. We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies.

218 EJACULATE TRAITS OF THE NAMIBIAN CHEETAH (ACINONYX JUBATUS)—INFLUENCE OF ANIMAL AGE, SEASON, AND CAPTIVITY

2006 ◽  
Vol 18 (2) ◽  
pp. 217
Author(s):  
A. E. Crosier ◽  
L. L. Marker ◽  
J. G. Howard ◽  
B. S. Pukazhenthi ◽  
J. N. Henghali ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Large Scale ◽  
Sperm Concentration ◽  
Ex Situ ◽  
Motile Sperm ◽  
Genetic Management ◽  
Acinonyx Jubatus ◽  
Sperm Density ◽  
A Genome ◽  
Hot Dry Season

Wild cheetahs are threatened with extinction, and ex situ populations are not self-sustaining due to poor reproductive efficiency. Sperm cryo-preservation is a valuable tool for genetic management; however, increased knowledge of ejaculate traits is essential to improve cryopreservation protocols. The objective of this study was to characterize ejaculate traits of wild-born cheetahs in Namibia, Africa. Specifically, the influences of animal age, season and captive status on electroejaculate volume, sperm concentration, motility, forward progressive status (FPS scale 0-5, 5 = best), morphology, and acrosomal integrity were evaluated. Animal age was divided into categories: juvenile (14-24 mo; n = 16 males, 23 ejaculates), adult (25-120 mo; n = 76 males, 175 ejaculates), and aged (over 120 mo; n = 5 males, 5 ejaculates). Namibian seasons were divided into hot-wet (Jan-Apr), cold-dry (May-Aug) and hot-dry (Sep-Dec). Cheetahs were considered wild-caught (n = 29 males; 44 ejaculates) if trapped on farmland d30 days before semen collection. Raw ejaculates contained 69.0 � 1.1% motile sperm (mean � SEM) and 73.6 � 1.5% sperm with intact acrosomes. Overall, 18.4 � 0.9% of sperm were morphologically normal, with midpiece abnormalities being the most prevalent defects (?39%). To determine treatment differences, data were analyzed by General Linear Model procedures and means were separated with Duncan's multiple-range test. Juvenile cheetahs produced ejaculates with reduced (P < 0.05) sperm motility (56.7 � 3.3%) and FPS (2.9 � 0.1) compared to adult (69.8 � 1.4% and 3.4 � 0.1, respectively) and aged (78.9 � 6.7% and 3.7 � 0.3, respectively) animals. Ejaculates from juvenile animals also had reduced (P < 0.05) volume (0.69 � 0.3 mL) and fewer (P < 0.05) total motile sperm (7.1 � 9.3 � 106) compared to adult (2.2 � 0.1 mL and 42.3 � 4.1 � 106) and aged (2.3 � 0.6 mL and 23.5 � 20.0 � 106, respectively) males. For all ejaculates combined, seminal quality was poorest during the hot-dry season with lower (P < 0.05) sperm motility and intact acrosomes as well as an increased (P < 0.05) percent of sperm with head abnormalities. Ejaculates from captive cheetahs (n = 68 males, 159 ejaculates) had increased (P < 0.05) volume (2.0 � 0.2 mL) and intact acrosomes (80.1 � 3.6%), but lower (P < 0.05) sperm density (14.3 � 3.9 � 106/mL) than wild-caught animals (1.5 � 0.3 mL, 71.9 � 4.6%, and 24.1 � 5.1 � 106/mL, respectively). These are the first large-scale data acquired to examine the reproductive biology of male cheetahs in Namibia, including those recently captured from the wild. Results reveal that this species demonstrates seasonal and age-based variations in ejaculate quality, and that all individuals (including those recently derived from the wild) produce unusually high proportions of pleiomorphic spermatozoa. These data are being used to select the ideal donor age and season during which spermatozoa should be collected for addition to a genome resource bank, thereby enhancing effective genetic management for cheetahs propagated ex situ.

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