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.

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.

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.

Comparison of different sperm cryopreservation procedures on post-thaw quality and heterologous in vitro fertilisation success in the ocelot (Leopardus pardalis)

2007 ◽  
Vol 19 (5) ◽  
pp. 685 ◽  
Author(s):  
Monica A. Stoops ◽  
Jennifer B. Bond ◽  
Helen L. Bateman ◽  
Mark K. Campbell ◽  
Gregory P. Levens ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Concentration ◽  
Domestic Cat ◽  
Ex Situ ◽  
In Vitro Fertilisation ◽  
Sperm Cryopreservation ◽  
Leopardus Pardalis ◽  
Field Environment

Cryopreservation of spermatozoa from free-living ocelots (Leopardus pardalis) could benefit their conservation by facilitating gene flow between in situ and ex situ populations without requiring removal of additional cats from the wild. The objective of this study was to investigate three different methods of ocelot sperm cryopreservation to identify the most appropriate technique for use in a field environment. Male ocelots (n = 10), housed in North American zoos, were anaesthetised with tiletamine–zolazepam (7mg kg–1 bodyweight; i.m.) and subjected to a regimented electroejaculation procedure. Recovered semen was evaluated for sperm concentration, motility and morphology and processed for cryopreservation by three methods: (1) pelleting on dry ice, (2) freezing in straws over liquid nitrogen vapour; and (3) freezing in straws in a dry shipper. Frozen samples were thawed and assessed for post-thaw acrosome status, viability, motility over time and ability to fertilise viable domestic cat oocytes. Although several post-thaw sperm parameters varied (P < 0.05) among freezing methods, frozen–thawed ocelot spermatozoa from all treatments showed a similar (P > 0.05) capacity to bind, penetrate and fertilise viable domestic cat oocytes. These findings suggest that spermatozoa collected from male ocelots under field conditions may be frozen in straws either using liquid nitrogen alone or in a charged dry shipper to retain adequate functional competence after thawing for use with assisted reproductive procedures.

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.

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 Oviductal Extracellular Vesicles Improve Post-Thaw Sperm Function in Red Wolves and Cheetahs

2020 ◽  
Vol 21 (10) ◽  
pp. 3733 ◽  
Author(s):  
Marcia de Almeida Monteiro Melo Ferraz ◽  
Jennifer Beth Nagashima ◽  
Michael James Noonan ◽  
Adrienne E. Crosier ◽  
Nucharin Songsasen
Keyword(s):  
Extracellular Vesicles ◽  
Sperm Motility ◽  
Wildlife Conservation ◽  
Species Conservation ◽  
Genetic Material ◽  
Ex Situ ◽  
Sperm Function ◽  
Red Wolf ◽  
Cryopreserved Sperm

Artificial insemination (AI) is a valuable tool for ex situ wildlife conservation, allowing the re-infusion and dissemination of genetic material, even after death of the donor. However, the application of AI to species conservation is still limited, due mainly to the poor survival of cryopreserved sperm. Recent work demonstrated that oviductal extracellular vesicles (oEVs) improved cat sperm motility and reduced premature acrosomal exocytosis. Here, we build on these findings by describing the protein content of dog and cat oEVs and investigating whether the incubation of cryopreserved red wolf and cheetah sperm with oEVs during thawing improves sperm function. Both red wolf and cheetah sperm thawed with dog and cat oEVs, respectively, had more intact acrosomes than the non-EV controls. Moreover, red wolf sperm thawed in the presence of dog oEVs better maintained sperm motility over time (>15%) though such an improvement was not observed in cheetah sperm. Our work demonstrates that dog and cat oEVs carry proteins important for sperm function and improve post-thaw motility and/or acrosome integrity of red wolf and cheetah sperm in vitro. The findings show how oEVs can be a valuable tool for improving the success of AI with cryopreserved sperm in threatened species.

scholarly journals Foreword: A perspective on the role of emerging technologies for the propagation of companion animals, non-domestic and endangered species

2007 ◽  
Vol 19 (6) ◽  
pp. iii ◽  
Author(s):  
Monique C. J. Paris ◽  
Gabriela F. Mastromonaco ◽  
Damien B. B. P. Paris ◽  
Rebecca L. Krisher
Keyword(s):  
Endangered Species ◽  
Emerging Technologies ◽  
Assisted Reproductive Technologies ◽  
Companion Animals ◽  
Reproductive Technologies ◽  
Reproductive Physiology ◽  
In Vitro Fertilisation ◽  
Genetic Management ◽  
The Status

Assisted reproductive technologies (ART) have been used successfully in humans, domestic and laboratory species for many years. In contrast, our limited knowledge of basic reproductive physiology has restricted the application of ART in companion animal, non-domestic and endangered species (CANDES). Although there are numerous benefits, and in some cases a necessity, for applying ART for the reproductive and genetic management of CANDES, the challenges encountered with even the most basic procedures have limited the rate of progress. In this foreword we discuss the status of conventional ART, such as artificial insemination and in vitro fertilisation, as well as their benefits and inherent difficulties when applied to CANDES. It is upon these techniques, and ultimately our knowledge of basic reproductive physiology, that the success of emerging technologies, such as those described in this special issue, are dependent for success.

Slow cooling prevents cold-induced damage to sperm motility and acrosomal integrity in the black-footed ferret (Mustela nigripes)

2007 ◽  
Vol 19 (5) ◽  
pp. 652 ◽  
Author(s):  
R. M. Santymire ◽  
P. E. Marinari ◽  
J. S. Kreeger ◽  
D. E. Wildt ◽  
J. G. Howard
Keyword(s):  
Sperm Motility ◽  
Seminal Plasma ◽  
Artificial Insemination ◽  
Sperm Cryopreservation ◽  
Slow Cooling ◽  
Rapid Cooling ◽  
Mustela Nigripes ◽  
Factors Influencing ◽  
Rate Of Cooling ◽  
Acrosomal Integrity

The endangered black-footed ferret (Mustela nigripes) has benefited from artificial insemination; however, improved sperm cryopreservation protocols are still needed. The present study focused on identifying factors influencing gamete survival during processing before cryopreservation, including: (1) the presence or absence of seminal plasma; (2) temperature (25°C v. 37°C); (3) type of medium (Ham’s F10 medium v. TEST yolk buffer [TYB]); (4) cooling rate (slow, rapid and ultra-rapid); and (5) the presence or absence of glycerol. Seminal plasma did not compromise (P > 0.05) sperm motility or acrosomal integrity. Sperm motility traits were maintained longer (P < 0.05) at 25°C than at 37°C in Ham’s or TYB, but temperature did not affect (P > 0.05) acrosomal integrity. Overall, TYB maintained optimal (P < 0.05) sperm motility compared with Ham’s medium, but Ham’s medium maintained more (P < 0.05) intact acrosomes than TYB. Slow cooling (0.2°C min–1) was optimal (P < 0.05) compared to rapid cooling (1°C min–1), and ultra-rapid cooling (9°C min–1) was found to be highly detrimental (P < 0.05). Results obtained in TYB with 0% or 4% glycerol were comparable (P > 0.05), indicating that 4% glycerol was non-toxic to ferret sperm; however, glycerol failed to ameliorate the detrimental effects of either rapid or ultra-rapid cooling. The results of the present study demonstrate that the damage observed to black-footed ferret spermatozoa is derived largely from the rate of cooling.

Integrating fertility preservation and cryo-banking into the conservation of rare and endangered deer species

2020 ◽  
Vol 60 (10) ◽  
pp. 1227
Author(s):  
P. Comizzoli
Keyword(s):  
Fertility Preservation ◽  
Reproductive Traits ◽  
Habitat Destruction ◽  
Ex Situ ◽  
In Vitro Fertilisation ◽  
Deer Species ◽  
Frozen Semen ◽  
Rare And Endangered

More than 50 deer species live in diverse ecosystems around the world. Unfortunately, most of them are threatened or endangered because of over-hunting, poaching or habitat destruction. Protection of wild populations (in situ) and management of animal collections in zoos and breeding centres (ex situ) are complementary conservation efforts relying on multidisciplinary approaches. Reproductive biology of deer species is one of the critical areas that still needs to be thoroughly studied to ensure the success of in situ or ex situ programs. Interestingly, there is a vast diversity in reproductive traits within the deer family (from anatomy to breeding-season patterns). On the basis of this fundamental knowledge, adapted reproductive biotechnologies have been developed to enhance reproduction and preserve fertility of individuals. Early works on artificial insemination (AI), in vitro fertilisation (IVF), and germplasm freezing in the more common red deer, sika deer and white-tailed deer have been highly inspiring to projects aiming at saving endangered deer species. A few fawn births following AI or IVF using frozen semen have been reported in wild species (e.g. Eld’s deer, Rucervus eldii thamin); however, assisted reproductive techniques and cryo-banking are currently not integrated into the management of rare and endangered populations. Knowing that many deer populations are rapidly declining in situ and ex situ, there is now an urgent need for better strategies and more fertility preservation options. The objectives of the present article are to review (1) existing reproductive biotechnologies to preserve fertility of different deer species and (2) how to integrate these approaches into the management of rare and endangered populations to address conservation issues.

116 IMPROVED SURVIVAL BY CRYOPRESERVING RHESUS MACAQUE (MACACA MULATTA) SPERMATOZOA WITH DIRECTIONAL FREEZING TECHNIQUE

2010 ◽  
Vol 22 (1) ◽  
pp. 217 ◽  
Author(s):  
W. Si ◽  
Y. Lu ◽  
X. He ◽  
S. Ji ◽  
Y. Niu ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Cooling Rate ◽  
Sperm Motility ◽  
Genetically Engineered ◽  
Human Diseases ◽  
Slow Cooling ◽  
Cooling Rates ◽  
Important Species ◽  
Directional Freezing ◽  
Acrosomal Integrity

A significant increase in nonhuman primate models of human diseases will be expected in the near future since the successes in production of genetically engineered rhesus monkey models of human diseases. Sperm banking can provide an effective way to preserve valuable genetic resources. Our objective was to (1) develop a protocol using directional freezing technique (DFT) for rhesus monkey spermatozoa cryopreservation, which allows precise control of the velocity and the morphology of the ice-front propagation by transferring the tubes loaded with 2 mL sperm samples at a controllable velocity through two separate chambers with controllable temperature settings, and (2) achieve survival rate that was higher than that achieved with conventional freezing technique (CFT), by which sperm samples were cryopreserved in 0.25 mL straws with liquid nitrogen vapor in a styrofoam box. Sperm motility, acrosomal integrity, and in vitro fertilization (IVF) assay were used to assess the function of frozen-thawed spermatozoa. Data were analyzed by ANOVA and Fisher protected LSD test. Experiment 1 was aimed at optimizing the cooling rate using DFT. Tubes were frozen using the multi-thermal gradient freezing device (MTG 516, Harmony CryoCareTM, IMT Ltd.) at fast (16°C/min), medium (12°C/min), and slow (7°C/min) cooling rates, which corresponded to the transferring velocities (2.5, 1.5, and 0.5 mm s-1, respectively). The results showed that spermatozoa frozen at fast and medium cooling rates showed significantly higher frozen-thawed motility than those frozen at slow cooling rate (61% and 59% v. 50%, P < 0.05). However, no difference was observed on sperm acrosomal integrity among the experimental groups (84, 80, and 78%, respectively, P > 0.05). The purposes of Experiment 2 were determined to examine if using DFT at the optimized cooling rate (12°C/min) can improve the cryo-survival of rhesus monkey spermatozoa compared with CFT. Our results showed that spermatozoa cryopreserved by using DFT achieved significantly higher frozen-thawed sperm motility that those cryopreserved by using CFT (64 v. 54%, P < 0.05). However, no difference was observed on acrosomal integrity between spermatozoa cryopreserved by DFT and CFT (84 and 83%, respectively; P > 0.05). The function of spermatozoa cryopreserved by using DFT was further evaluated by IVF. Females were treated with rhFSH twice-daily for 8 days after the onset of menses and following a treatment of hCG injection on Day 9. Cumulus-oocyte complexes were collected by laparoscopic follicular aspiration 32 h later. Of the inseminated oocytes, 79% were fertilized and 90 and 53% of the resulting zygotes developed into 2-cell and blastocysts, respectively. The fertilization rate was lower and the blastocyst rate was slightly higher than our previous report when fresh spermatozoa were used for IVF (94 and 52%, respectively). Our results indicate that spermatozoa of rhesus monkeys can be effectively cryopreserved using DFT in large volume. This finding provided a new and effective way for genetics preservation purposes in this important species.

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