199 IN VITRO EMBRYO PRODUCTION IN THE CLOUDED LEOPARD (NEOFELIS NEBULOSA)

2010 ◽  
Vol 22 (1) ◽  
pp. 258 ◽  
Author(s):  
C. E. Pope ◽  
M. C. Gómez ◽  
B. L. Dresser
Keyword(s):  
Oocyte Retrieval ◽  
Ovarian Response ◽  
Ovarian Activity ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Total N ◽  
Clouded Leopard ◽  
Vitro Production

The clouded leopard, a spotted, mid-sized cat native to southeast Asia, is classified as Endangered by the U.S. Fish and Wildlife Service. The Species Survival Plan (SSP) program has designated that clouded leopards in North American zoological institutions are a research population with the focus of enhancing management and developing assisted reproductive techniques. In this study, we examined (1) ovarian response to exogenous gonadotropin stimulation, (2) in vitro production of embryos by IVF and intracytoplasmic sperm injection (ICSI), and (3) in vivo developmental ability of in vitro-derived embryos. Eight females at Audubon Center for Research of Endangered Species (ACRES) in New Orleans, LA, were used as oocyte donors and embryo recipients. During non-estrual, but otherwise unknown, stages of the estrous cycle, females 4 to 10 years old at our first treatment were administered a total of 15 (n = 8) or 20 (n = 7) IU of porcine FSH (i.m; Sioux Biochemical Co., Sioux City, IA) daily for 4 days. On Day 5, 10 (n = 8) or 15 (n = 7) IU of porcine LH (i.m; Sioux Biochemical Co.) was given and laparoscopic oocyte retrieval (LOR) was done 24 h later. One, 2, or 3 LOR were performed on 3, 3, and 2 females, respectively (total n LOR = 15), 10 (67%) of which were done on females that were 8 to 12 years of age. A total of 176 pre-ovulatory oocytes (mean = 14.7; range = 3-31) were recovered from 12 LOR performed on 6 females. Two females, a 5-year-old and an 11-year-old, did not respond to gonadotropin treatment. Of 5 females with ≥2 LOR, an average of 16.2 and 14.6 oocytes were recovered at the first and second LOR, respectively. Luteal tissue was present on the ovaries at 3 of 4 LOR performed during January-April as compared with 0 of 11 LOR performed from June-December. Semen was obtained by electroejaculation of 3 males (6, 9, and 11 years of age) and used after cryopreservation [TEST-yolk (media prepared with egg yolk, TES, and Tris) + 6% glycerol] or after storage at 4°C for 24 h (cooled). In vitro production, cryopreservation, and transfer of embryos were done as described by Pope et al. (2006 Theriogenology 66, 1518-1524). Cleavage frequency was 43% overall, 55% (64/117) after IVF, and 20% after ICSI with frozen (10/43) or cooled (2/16) sperm. After IVF with frozen sperm, cleavage rate was 63% (48/76) v. 39% (16/41) with cooled sperm. On Days 5, 57, 26, and 17% of embryos were morula (> 16 cells), early morula (≈16 cells) and pre-morula (< 16 cells), respectively. Forty-four Day 5 embryos were cryopreserved at a slow, controlled rate, 24 of which were thawed and transferred by laparotomy to the uteri of 3 Day 5 gonadotropin-treated recipients (7-9 embryos/female). Similarly, 28 Day 5 fresh embryos were auto-transferred to 3 Day 5 recipients (5-15 embryos/female). Most embryos transferred were morulae (83%) produced by IVF using frozen sperm (71%). None of the recipients established pregnancies, a failure that, undoubtedly, was multifaceted. Age may have been a factor because most recipients were 8 to 11 years old. Nonetheless, we have (1) shown that clouded leopard females are responsive to exogenous gonadotropins, (2) produced embryos in vitro, both by IVF and ICSI, using both cryopreserved and cooled sperm, and (3) provided further evidence of seasonality in ovarian activity.

122 BIRTH OF BLACK-FOOTED CAT KITTENS AFTER TRANSFER OF CRYOPRESERVED EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH CRYOPRESERVED SPERM

2012 ◽  
Vol 24 (1) ◽  
pp. 173 ◽  
Author(s):  
C. E. Pope ◽  
M. C. Gómez ◽  
C. Dumas ◽  
R. A. MacLean ◽  
E. Crichton ◽  
...  
Keyword(s):  
Oocyte Retrieval ◽  
Ovarian Response ◽  
In Vitro Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Cryopreserved Sperm ◽  
Transfer Procedures ◽  
Cryopreserved Embryos

The black-footed cat (Felis nigripes), a diminutive spotted cat whose native habitat is arid grasslands in South Africa, Namibia and Botswana, is classified as vulnerable by the International Union for Conservation of Nature and is listed as CITES Appendix I. They are perhaps the rarest of the African cats and their status is threatened by habitat deterioration and poisoning from ingestion of baited carcasses intended for other species of cats. Here, we examined (1) ovarian response of black-footed cat females to exogenous gonadotropin stimulation, (2) in vitro production of embryos by IVF with cooled vs cryopreserved sperm and (3) in vivo developmental ability of in vitro–derived embryos. Six females, 1.5 to 2.5 years of age at first treatment, were administered a total of 3.0 to 3.6 IU of porcine FSH (IM; Sioux Biochemical Co., Sioux City, IA) daily over 4 days. On Day 5, 3.0 (n = 12) or 5.0 (n = 2) IU of porcine LH (IM; Sioux Biochemical Co.) was given and laparoscopic oocyte retrieval (LOR) was done 24 h later. One, two, three, or four LOR were done on 1, 3, 1 and 1 females, respectively (total = 14 LOR). The average age at LOR was 3.3 years (range = 1.5–7.5 years). Semen was obtained by electroejaculation of 3 males (1.5, 6.0, 7.5 years). Anaesthesia for LOR and electroejaculation was induced and maintained, after intubation, with 5 and 2.5% isoflurane, respectively. Sperm samples were used after storage at 4°C for 24 h (TEST yolk, TY) or after cryopreservation (TY + 6% glycerol). Luteal tissue was present on the ovaries at 4 of 5 LOR done during January to May as compared with none of 9 LOR done from June to December. Of 165 oocytes (mean = 11.8) recovered, 38/54 (70%) and 50/106 (47%) underwent cleavage after IVF with cooled or cryopreserved sperm, respectively (P < 0.01, chi-square). None of 5 oocytes cleaved after intracytoplasmic sperm injection with cryopreserved sperm. Procedures for in vitro embryo production were as described previously (Gómez et al. 2006 Theriogenology 66, 72–81; Pope et al. 2006 Theriogenology 66, 1518–1524). Four laparoscopic oviducal embryo transfer procedures were done on Day 1: 2 recipients received fresh Day 2 embryos (n = 5, 8) and 2 recipients received embryos that had been cryopreserved on Day 1 (n = 6) or 2 (n = 8) at a slow, controlled rate in 1.4 M of propylene glycol/0.125 M of sucrose/10% dextran 70. Each recipient (1.75 to 4.5 years) had undergone LOR on Day 0 (5–19 oocytes recovered). Upon ultrasonographic examination on Day 50, a 2.3-year-old recipient of cryopreserved embryos was determined to be pregnant. She delivered 2 live male kittens, without assistance, on Day 69. When first examined at 15 days of age, the kittens weighed 156 and 198 g. At 5 months, their weights were 1.62 and 1.81 kg. The sperm sample used to produce the embryos (in 2005) that resulted in the births of kittens (in 2011) was collected from a male at the Henry Doorly Zoo, Omaha, NE (in 2003), extended and transported overnight at 4°C to New Orleans, LA, before cryopreservation. In summary, we have further demonstrated that assisted reproductive technology can be used for conservation of rare and vulnerable small felids.

scholarly journals MATURAÇÃO E FECUNDAÇÃO IN VITRO DE OÓCITOS BOVINOS EM TUBOS PREVIAMENTE GASEIFICADOS E MANTIDOS EM BANHO-MARIA

2002 ◽  
Vol 7 (2) ◽  
Author(s):  
M. KURTZ FILHO ◽  
L. M. SILVA ◽  
B. MOREIRA ◽  
D. S. BRUM ◽  
F. G. LEIVAS ◽  
...  
Keyword(s):  
Water Bath ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Embryo Production ◽  
Vitro Production

A produção in vitro (PIV) de embriões bovinos alcançada com vacas de matadouros ou de aspiração folicular in vivo (OPU) é uma prática cada vez mais difundida e a sua simplificação poderia baixar os custos de produção. O objetivo desta pesquisa foi comparar a produção in vitro de embriões em estufa com temperatura, umidade relativa e atmosfera controlada (controle), com tubos de poliestireno gaseificados e mantidos em banho-maria (tratamento). Oócitos obtidos de ovários de vacas abatidas foram maturados in vitro em TCM- 199 modificado com 25mM de N-2-hidroxietilpiperazina-N -2-ácido etanosulfônico (HEPES); 0,025mg/ml de piruvato de sódio, 0,01UI de rFSHh/ml, 0,5µg/ml de LHs e 10% de soro de vaca em estro. Na fecundação in vitro utilizou-se Talp-Fert com 0,06mg/ml de albumina sérica bovina, 0,022mg/ml de piruvato de sódio e 10µg/ml de heparina. O cultivo foi conduzido em placas de 4 poços em SOF com 5% de soro de vaca em estro, 20µl/ml de aminoácidos essenciais e 10µl/ml de aminoácidos não essenciais, sob óleo mineral, em estufa com atmosfera de 5% de CO2, umidade saturada e 39°C, por 9 dias. Na maturação não houve diferença (P>0,05) entre o tratamento e o controle. Porém, a maturação e a fecundação ou somente a fecundação in vitro em tubos mantidos em banho-maria não demonstrou ser uma alternativa recomendada para a produção de embriões bovinos. In vitro maturation and fertilization of bovine oocytes in tubes previously gasified kept in water bath Abstract In vitro bovine embryo production either obtained from oocytes of slaughtered cows or in vivo follicular aspiration (OPU) is a well-known technique and it’s simplification might reduce the cost of embryo production. The aim of this study was to compare the cleavage rate and embryo development of the in vitro production of bovine embryos using standard culture system (temperature, gas phase and controlled humidity) versus gasified polystyrene tubes kept in water bath. Oocytes obtained from ovaries of slaughtered cows were in vitro maturated in TCM- 199’modified with 25 mM of N-2-hidroxyethylpiperazine-N’-2-ethanosulfonic acid (HEPES); containing 0.01UI rFSHh/ml and 0.5µg/ml LHs, 0.025mg/ml sodium pyruvate and 10% estrous cow serum. The in vitro fertilization was carried out in Talp-Fert containing 0.06mg/ml BSA, 0.022mg/ml sodium pyruvate and 10µg/ml heparin. The culture was performed in SOF medium with 20µl/ml essential aminoacids, 10µl/ml, non-essential aminoacids and 5% estrous cow serum, with oil overlay, in 4 well dishes and incubated with 5% CO2, maximum humidity at 39°C, for 9 days. The results of this study showed no difference (P>0.05) between the treatment and control groups during the maturation process. However, the maturation and fertilization or only the fertilization in tubes do not represent a viable alternative for the in vitro production of bovine embryos.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.

scholarly journals Reprogrammed Cell?based Therapy for Liver Disease: From Lab to Clinic

2017 ◽  
Vol 1 (1) ◽  
pp. 20-28 ◽  
Author(s):  
Amir Mehdizadeh ◽  
Masoud Darabi
Keyword(s):  
Cellular Reprogramming ◽  
Gene Correction ◽  
In Vitro Production ◽  
Efficient Treatment ◽  
Cell Based Therapy ◽  
Number Of Patients ◽  
End Stage ◽  
Vitro Production

A large number of patients are affected by liver dysfunction worldwide. Liver transplantation is the only efficient treatment in a variety of enduring liver disorders including inherent and end-stage liver diseases. The generation of human functional hepatocytes in high quantities for liver cell therapy is an important goal for ongoing therapies in regenerative medicine. Reprogrammed cells are considered as a promising and unlimited source of hepatocytes, mainly because of their expected lack of immunogenicity and minimized ethical concerns in clinical applications. Despite gained advances in the reprogramming of somatic cells to functional hepatocytes in vitro, production of primary adult hepatocytes that can proliferate in vivo still remains inaccessible. As part of efforts toward translation of cell reprogramming science into clinical practice, more careful cell selection strategies should be integrated into improvement of dedifferentiation and redifferentiation protocols, especially in precision medicine where gene correction is needed. Furthermore, advances in cellular reprogramming highlight the need for developing and evaluating novel standards addressing clinical research interests in this field.

86 Comparison of in vivo and in vitro embryo production in Bonsmara donors

2019 ◽  
Vol 31 (1) ◽  
pp. 168
Author(s):  
B. H. Bernal ◽  
J. L. Barajas ◽  
J. A. Ortega ◽  
A. Cedeño ◽  
S. Andrada ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Gonadotropin Releasing Hormone ◽  
P Value ◽  
In Vitro Production ◽  
Embryo Production ◽  
Releasing Hormone ◽  
Gradient Technique ◽  
Vitro Production

A retrospective analysis of embryo production records from 2013 to 2017 was carried out to evaluate the in vivo and in vitro production (IVP) of embryos in donors of the Bonsmara breed (i.e. tropically adapted Bos taurus). Only donors with production records of both in vivo and in vitro embryos during the same period were used. A total of 127 superovulations and ova/embryo collections of 19 donors were evaluated. The donors were superstimulated with the following protocol: on Day 0 they received a device with 1g of progesterone (DIB, Zoetis, Argentina), 50mg of rogesterone (Progestar, Zoetis), and 5mg of oestradiol-17β (17ßOestradiol, Rio de Janeiro, Argentina) or 2mg of oestradiol benzoate (Gonadiol, Zoetis) intramuscularly (IM) at the same time. Superstimulatory treatments were initiated on the morning of Day 4 with Folltropin-V (Vetoquinol, France; total dose=240 to 340mg IM) in twice-daily decreasing doses over 4 days. All donors received 2 IM injections of 500µg of cloprostenol (Ciclase DL, Zoetis) on the morning and afternoon of Day 6 and; the intravaginal devices were removed on the morning of Day 7 and 100µg of Gonadorelin (gonadotropin-releasing hormone, Gonasyn gdr, Zoetis) was given on the morning of Day 8. Donors were inseminated using semen from 9 Bonsmara bulls, 12 and 24h after gonadotropin-releasing hormone. On Day 15, ova/embryos were collected and classified according to IETS standards. A total of 89 follicular aspirations (ovum pickup) of 19 donors for IVP were evaluated. The ovum pickups were performed at random stages of oestrous cycle, without superstimulation or other hormone treatments. A total of 1109 viable oocytes (12.5±0.9 per ovum pickup) were collected and matured for 24h in 100-µL drops of maturation medium (TCM-199, supplemented with hormones) under mineral oil and incubated at 38.5°C in 5.5% CO2 and humidity at saturation. Fertilization was performed using 3 Bonsmara bulls that were also used for in vivo embryo production. Viable sperm were obtained using the percoll gradient technique (45-90%). The sperm pellet was dissolved in TL-Sperm, centrifuged, and then diluted to a final concentration of 1.5×106 sperm/mL. Zygotes were stripped and placed in drops of 100µL of SOF medium supplemented with 0.4% BSA under oil at 38.8°C, 5.5% CO2, 7% O2, and humidity at saturation for 7 days. The culture medium was renewed on Days 3 and 5. The data were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA), a P-value &lt;0.05 was considered significant. The mean (±standard error of the means) number of CL, ova/embryos collected, fertilized ova, and transferable embryos were 12.9±0.6, 8.8±0.6, 6.6±0.5, and 4.7±0.4, respectively. A total of 662 oocytes (66.3±2.4%) cleaved 48h post-IVF. On Day 7, an average of 4.4±0.3 embryos were produced. No differences were detected in the number of transferable embryos produced in vivo v. those produced in vitro. Furthermore, no significant differences were found between the techniques or bulls on the proportion of embryos produced in relation to the ova/embryos or oocytes obtained (in vivo 51.5±3.2% v. in vitro 42.9±2.5%). In conclusion, the in vivo and in vitro production of embryos are both effective alternatives to increase the number of offspring from valuable Bonsmara donors.

Sign in / Sign up

Export Citation Format

Share Document