In vitro embryo production and embryo transfer in domestic and non-domestic cats

Over a 5-year interval, multiple laparoscopic oocyte retrievals were done in fishing cats (Prionailurus viverrinus), caracals (Caracal caracal) and domestic cats after ovarian stimulation with gonadotropins. From 21 retrievals in five fishing cats, 579 preovulatory oocytes (mean = 27.6) were recovered and 348 embryos were produced in vitro (mean = 16.6). A total of 452 preovulatory oocytes (mean = 25.1) were recovered from 18 of 24 retrievals in six caracals and 297 (mean = 16.6) embryos were produced. An additional 16 caracal embryos (19%) were produced after in vitro maturation of 83 oocytes, 59 of which came from six retrievals producing only immature oocytes. The presence of corpora lutea at oocyte retrieval occurred in each species (1) at a similar frequency (33%) and (2) more frequently during January through May (11 of 15 retrievals) than during the latter half of the year (4 of 30 retrievals). Of the 12 embryo transfer procedures done in fishing cats, one pregnancy (8%) was obtained and one live kitten born after the auto-transfer of 10 Day-6 embryos. In caracals, a total of 46 Day-4 or Day-5 embryos were auto-transferred to six recipients, one of which delivered two live kittens. Then, 109 caracal embryos were cryopreserved before thawing and transferring to nine recipients (mean = 12.1) on Days 5 or 6. From three pregnancies established (33%), a total of three kittens were born. Two to six gonadotropin treatments/oocyte retrievals were done in domestic cats during 1999 through 2003; an average of 24.9, 23.5, 22.0, 23.1, 23.5 and 40.9 oocytes (P > 0.05) were recovered at the first through the sixth treatment cycles from 138, 138, 97, 49, 22, and seven retrievals, respectively.     

In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach

Development of new technology related to in vitro embryo production has allowed for the commercial use of this method of reproduction. In the present work, we evaluate the efficiency of this technology compared with conventional embryo production based on results obtained with a standard procedure, including the sexing of embryos. The donor animals were mature nonlactating dairy cows (n = 92) kept under a constant environment and feeding program in an ET center. Ultrasound guided transvaginal ovum pick-up following 48 h pre-treatment with FSH has been used for the IVF-IVC protocol. A total of 437 oocyte recovery sessions performed on 92 cows yielded 4145 oocytes, which were used in an IVF-IVC protocol. Using the conventional approach, 156 embryo collections on 49 cows yielded 1652 ova and embryos. All Quality 1 and 2 embryos were sexed by a PCR procedure, and embryos of the desired sex were transferred to synchronized recipients located at the center. The results obtained in the IVF protocol showed that 4 oocyte collections per cow performed within 60 d, yielded 38 oocytes, which resulted in 18.8 viable embryos, of which 7.05 were female. After transfer of the female embryos, an average of 3.8 recipients were pregnant at 60 d. One embryo collection under the conventional approach yielded an average of 1.2 female pregnancies, which was confirmed during the same 60-d time period. These results indicate that IVF procedures can effectively replace conventional embryo production methods when a predetermined number of pregnancies of known sex are needed within a short period of time.     

In vivo and in vitro embryo production in goats

Assisted reproductive technologies (ART) such as artificial insemination (AI) and multiple ovulation and embryo transfer (MOET) have been used to increase reproductive efficiency and accelerate genetic gain. The principal limitations of MOET are due to variable female response to hormonal treatment, fertilization failures and premature regression of Corpora luteum. The in vitro production (IVP) of embryos offers the possibility of overcoming MOET limitations. The method of IVP of embryos involves three main steps: in vitro maturation of oocytes (IVM), in vitro fertilization of oocytes (IVF) with capacitated sperm and in vitro culture (IVC) of embryos up to blastocyst stage. Recovering oocytes from live selected females by laparoscopic ovum pick-up (LOPU) and breeding prepubertal females by juvenile in vitro embryo technology (JIVET) will allow a greater production of valuable goats. Also, IVP of goat embryos will provide an excellent source of embryos for basic research on development biology and for commercial applications of transgenic and cloning technologies. Different protocols of IVP of embryos have been used in goats. However oocyte quality is the main factor for embryos reaching blastocyst stage from IVM/IVF/IVC oocytes. One of the principal determinant factors in the results of blastocyst development is the age of the oocyte donor females. In goats, oocytes from prepubertal and adult females do not show differences in in vitro maturation and in vitro fertilization; however the percentage of oocytes reaching blastocyst stage ranges from 12 to 36% with oocytes from prepubertal and adult goats, respectively.     

Application of assisted reproduction for population management in felids: the potential and reality for conservation of small cats

Assisted reproductive technology (ART), using the primary applied tools of AI, ET, and sperm and embryo cryopreservation, has been promoted over the past decades for its potential to conserve endangered wildlife, including felids. However, if the goal is efficient, consistent production of viable offspring for population management, then the 'potential' of ART has yet to become 'reality' for any non-domestic cat species. For the five small-sized felids (i.e., Brazilian ocelot, fishing cat, Pallas' cat, Arabian sand cat, black-footed cat) managed by Species Survival Plans (SSPs) in North American zoos, achieving this potential may be an absolute necessity if genetically viable captive populations are to be maintained into the next century. Modeling programs suggest that current SSP populations are not sustainable without periodic introduction of new founders and improved demographic parameters, including longer generation intervals and larger population sizes. ART provides the means to address each of these management challenges. In each small cat SSP species, fecal hormone metabolite assays and seminal analysis have proven useful for characterizing basal reproductive parameters, a necessary prerequisite to developing ART. Of the five SSP species, ART has been used to produce living offspring only in the ocelot, including after AI with frozen-thawed spermatozoa and following transfer of frozen-thawed IVF embryos. The true efficacy of these techniques, however, is still unknown. To improve the applicability of ART for population management, priorities for immediate research include further investigation of ovarian stimulation protocols, sperm and embryo cryopreservation methods, embryo culture systems, and fetal and neonatal viability following ART.     

Influence of oocyte donor on in vitro embryo production in buffalo

The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n = 40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A + B COC recorded in each 4-PS period had great repeatability (r = 0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A + B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r = 0.60; P < 0.001), SFL (r = 0.68; P < 0.001), COC (r = 0.48; P < 0.01) and Grade A + B COC (r = 0.40; P < 0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P < 0.05) number of TFL (8.3 ± 0.9 compared with 5.6 ± 0.7) and SFL (7.3 ± 0.3 compared with 3.8 ± 0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r = 0.80; P < 0.05) and COC (r = 0.76; P < 0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.     

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.     

Overall efficiency of in vitro embryo production and vitrification in cattle

In 5 replicates a total of 719 immature oocytes recovered from 94 slaughterhouse-derived bovine ovaries were matured and fertilized in vitro, then cultured for 7 to 9 d on a granulosa cell monolayer in TCM 199 supplemented with calf serum. Of 338 blastocysts (47% of oocytes cultured), 301 were vitrified in Hepes/bicarbonate buffered TCM-199 medium, 20% calf serum and dimethylsulfoxide and ethylene glycol as the cryoprotectants. After thawing in 1 M sucrose and subsequent culture in vitro, 237 (79%) of the blastocysts re-expanded and 177 (59%) hatched. Re-expansion and hatching rates differed between the blastocysts vitrified on Day 7 and Day 8 (84 and 69% vs 70 and 41%, respectively). We conclude that the applied methods are relatively simple and inexpensive to use, with an overall efficiency of the in vitro production/vitrification procedure being 1.9 hatched blastocyst/ovary. Therefore, this system seems suitable for large-scale production of cryopreserved bovine embryos for various purposes.     

Effect of follicular wave synchronization on in vitro embryo production in heifers

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7 d, followed by the administration of 150 μg d-cloprostenol. On Day 7, all follicles >3 mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14 d. Each experiment had three treatment groups. In Experiment 1 (n = 12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28 d. Heifers from Group 1X-EB also received 2 mg EB i.m. immediately after each OPU session. In Experiment 2 (n = 11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5 mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28 d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.     

Seasonality and reproduction in wild-living cats in Scotland

The definition of the wildcatFelis silvestris Schreber, 1775 in Scotland is contentious, in light of long-term interbreeding with domestic catsF. catus Linnaeus, 1758. Two morphological groupings (Group 1 and Group 2) have previously been proposed to explain the variation found in wild-living cats in Scotland, with Group 1 cats closest to wildcats and Group 2 cats to domestic cats. Data from the reproductive tracts of 185 wild-living cat carcasses and evidence of reproductive activity in 31 live cats were analysed in order to compare reproductive activity between the morphological groups, and in relation to seasonality and existing data on wildcats and domestic cats. For males, Group 2 cats had a greater mean relative testes size than Group 1 cats. Estimated from corpora lutea, there was a suggestion that Group 1 females showed more seasonality in oestrous than Group 2 cats. In all wild-living cats, the mean litter size was 4.3 and estimated birth dates were throughout the year, but least in winter. A high number of pseudopregnancies were recorded. The results were consistent with the hypothesis that Group 2 cats are closer to domestic cats. However, the variation observed in the sample of wild-living cats reported here, suggested that reproduction was neither strictly seasonal nor outside the range observed in some feral cat populations.     

Charles Thibault and assisted reproduction in France

Charles Thibault was liked by French gynaecologists. There was not a year that Charles Thibault did not attend clinician gynaecology conferences. He made great strides in research on in vitro fertilisation, being the first to perform in vitro fertilised (IVF) oocyte transfers in rabbits. Later, in 1978 the first human pregnancy following IVF was achieved in the UK when Louise Brown was born. In 1980, two French teams,one at the Sèvres hospital and the other at the Clamart University Teaching Hospital, carried out egg retrievals in patients with natural cycles, after determination of the urinary LH peak, under general anaesthesia and by laparoscopy. The Clamart team developed LH SIR, which enabled a more accurate determination of the ideal time for egg collection. In 1983, the same team reported the first ambulatory oocyte retrievals by ultrasound, under local anaesthesia. This new technique did not require general anaesthesia. Finally, in 1983, the rate of births, per transfer, for the Sèvres team rose to 5.31%. 1984 showed considerable improvement: 13.83%. The first step in establishing IVF in France was completed with the Carghese symposium, in September 1984, where Charles Thibault pleaded for animal experimentation before human clinical trials. It was only later that ART developed significantly, necessitating a legislative framework and organisations such as GEFF and FIVNAT.     

Review: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods

In vitro production (IVP) of embryos and associated technologies in cattle have shown significant progress in recent years, in part driven by a better understanding of the full potential of these tools by end users. The combination of IVP with sexed semen (SS) and genomic selection (GS) is being successfully and widely used in North America, South America and Europe. The main advantages offered by these technologies include a higher number of embryos and pregnancies per unit of time, and a wider range of potential female donors from which to retrieve oocytes (including open cyclic females and ones up to 3 months pregnant), including high index genomic calves, a reduced number of sperm required to produce embryos and increased chances of obtaining the desired sex of offspring. However, there are still unresolved aspects of IVP of embryos that limit a wider implementation of the technology, including potentially reduced fertility from the use of SS, reduced oocyte quality after in vitro oocyte maturation and lower embryo cryotolerance, resulting in reduced pregnancy rates compared to in vivo–produced embryos. Nevertheless, promising research results have been reported, and work is in progress to address current deficiencies. The combination of GS, IVP and SS has proven successful in the commercial field in several countries assisting practitioners and cattle producers to improve reproductive performance, efficiency and genetic gain.     

In vitro manipulation of nonhuman primate gametes for embryo production and embryo transfer.

Since nonhuman primates are closely related to humans and share many physical similarities, they are important for use in research areas such as human infectious diseases, reproduction, physiology, endocrinology, metabolism, neurology and longevity. To develop and maintain these animals, we must establish techniques for in vitro manipulation of spermatozoa and eggs. For a decade my research group has been conducting basic research to establish embryo manipulation techniques and to clarify the reproductive phenomena in nonhuman primates. This article summarizes the past research on in vitro manipulation of nonhuman primate gametes, from collection of reproductive cells and in vitro fertilization to the birth of offspring after embryo transfer, as well as the current status of these research areas. The studies summarized here will directly lead to the development of standard techniques for practical and comprehensive use in nonhuman primates.     

Rethinking in vitro embryo culture: new developments in culture platforms and potential to improve assisted reproductive technologies

The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future.     

Anthelmintics for dogs and cats

As the dog and cat anthelmintic market is small, fragmented and dominated by old-fashioned narrow spectrum products, there is little incentive for the pharmaceutical companies to invest major research resources in this area. Development of new products will always be of relatively low priority and progress will be much slower than is the case with ruminant anthelmintics. Some successes since ICOPA V can however be claimed. The previously reported cesticidal properties of praziquantel have been put to good use and have contributed to the progress made in many hydatid control programmes. Ivermectin shows promise as an alternative heartworm prophylactic and the applications for multiple dose therapy with the benzimidazoles continue to increase. In the latter case, much still needs to be done to explore the full potential of this chemical group and to define optimum dosage regimes for both broad spectrum therapy and for the prophylaxis of T. cati and A. caninum infections. It is to be hoped that the increasing awareness of the general public concerning the role of worm control in the broader concept of responsible pet ownership will create a greater demand for expert advice and more effective chemotherapy.     

Artificial reproduction of the Yangtze field vole: in vitro embryo development and fertilization with fresh and freeze-thawed sperm

Several research groups are using the Yangtze field vole as a model for studying schistosome infection, but relatively little is known about the species's reproductive physiology. The authors examined the vole's in vivo and in vitro embryonic development as well as the efficacy of in vitro fertilization using either fresh or cryopreserved sperm to breed these rodents.     

In vitro embryo production in buffalo species: state of the art.

In the last several years, there has been an increasing interest in in vitro embryo production (IVEP) technologies for faster propagation of superior germplasm in buffalo because of the low efficiency of superovulation (SO) and embryo transfer (ET) programs in this species. Although the IVEP efficiency has improved, embryo yield and development to term are still very low. This paper reviews the progress made in optimizing the IVM, IVF, and IVC systems. It also highlights the importance of embryo cryopreservation, which might critically contribute to the diffusion of ET procedures in the field. The acquisition of more information on embryo physiology, metabolism, and culture requirements in this species is critical to optimize the efficiency of advanced reproductive strategies. Further studies are also needed to improve the cryopreservation of IVEP embryos. The second part of the work underlines the potential impact of ovum pick-up (OPU) technique combined with IVEP on genetic improvement of buffalos. The OPU technique is a non-invasive and repeatable procedure for recovering large numbers of meiotically competent oocytes from antral follicles of live animals. Our experience, in buffalo, has demonstrated that OPU is superior to SO because it can yield more transferable embryos (TE) per donor on a monthly basis (2 TE vs 0.6, respectively). Therefore this technology has great potential to improve the genetic progress of buffalo through the maternal lineage.