Dissecting why superovulation and embryo transfer usually work on some farms but not on others

Bovine embryo transfer is a well-established commercial industry that is often associated with veterinary practices. Practitioners offering embryo transfer services may possess a very high standard of technical expertise; however, success in the production of embryos and the impregnation of recipients cannot be achieved unless the cattle are healthy and maintained in a well-managed cattle operation. In addition to appropriate gonadotropin treatments of donor cattle, the use of highly fertile semen, known to have been properly stored and handled is required for success. Recipient cattle must be managed with the same attention to detail as donors. Traditionally, PGF has been used for the synchronization of recipients. However, PGF is limited in its effectiveness early and late in the bovine estrus cycle. Recipient estrus synchronization with progesterone releasing intravaginal inserts has been successful and high pregnancy rates have resulted following embryo transfer.     

In vitro technologies related to pig embryo transfer

Embryo transfer in swine (ETS) has been used for commercial and breeding application only to a limited extent. However this technique is an essential prerequisite for the application of new reproductive techniques in pigs. This paper will give an overview on steps of pig embryo transfer including selection and stimulation of donor sows, recovery of embryos, embryo handling and the transfer of recovered embryos into recipients. Furthermore the current status and further application of ET related in vitro technologies in pig production are described.     

The current status of equine embryo transfer

The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulating mares. Because there is no commercially available hormonal preparation for inducing multiple ovulation in the horse, equine pituitary extract has been used to increase the number of ovulations in treated mares, but FSH of ovine or porcine origin is relatively ineffective in inducing multiple ovulation in the mare. Factors shown to affect pregnancy rates after embryo transfer include method of transfer, synchrony of the donor and recipient, embryo quality, and management of the recipient. One of the major improvements in equine embryo transfer over the last several years is the ability to store embryos at 5 degrees C and thus ship them to a centralized station for transfer into recipient mares. Embryos are collected by practitioners on the farm, cooled to 5 degrees C in a passive cooling unit and shipped to an embryo transfer station without a major decrease in fertility. However, progress in developing techniques for freezing equine embryos has been slow. Currently, only small, Day-6 equine embryos can be frozen with reasonable success. Additional studies are needed to refine the techniques for freezing embryos collected from mares 7 or 8 d after ovulation. Demand for the development of assisted reproductive techniques in the horse has increased dramatically. Collection of equine oocytes by transvaginal, ultrasound-guided puncture and the transfer of these oocytes into recipients is now being used to produce pregnancies from donors that had previously been unable to provide embryos. In vitro fertilization, however, has been essentially unsuccessful in the horse. One alternative to in vitro fertilization that has shown promise is intracytoplasmic sperm injection. However, culture conditions for in vitro-produced embryos appear to be inadequate. The continued demand for assisted reproductive technology will likely result in the further development of techniques that are suitable for use in the horse.     

The usefulness of a single measurement of insulin-like growth factor-1 as a predictor of embryo yield and pregnancy rates in a bovine MOET program

The objective was to determine if a single measurement of plasma insulin-like growth factor-1 (IGF-1) could predict the number of viable embryos obtained from donors and the likelihood of pregnancy in recipients in multiple ovulation and embryo transfer (MOET) programs in cattle. The embryo yields from 101 embryo recoveries were examined in maiden Holstein heifers (n=75) and multiparous Holstein cows (lactating cows n=20, dry cows n=6). Donors were super stimulated with FSH and embryo recovery was done non-surgically 7 days after artificial insemination. Embryos were classified according to the IETS criteria. Pregnancy rates in 100 maiden Holstein heifer recipients were analysed. Recipients were on day 7+/-1 of the estrous cycle at transfer. Pregnancy diagnosis was carried out at day 30 (PD 30) and rechecked at day 60 (PD 60) after transfer. Blood samples from coccygeal vessels taken at the time of embryo recovery (donors) and transfer (recipients) were analysed for IGF-1, insulin, beta-hydroxybutyrate (beta-OHB), non-esterified fatty acids (NEFA), urea and cholesterol. There was a negative correlation between the number of viable embryos and insulin (r=-0.33, P=0.025) in donor heifers. In donor cows, the number of viable embryos was correlated with IGF-1 (r=0.43, P=0.028) and cholesterol (r=-0.43, P=0.027). In recipients, PD30 and PD 60 were not affected by any of the circulating parameters analysed. Insulin, IGF-1 and cholesterol only explained 8.9, 13.9 and 15.8% of the variation in the production of viable embryos, respectively. Several factors affect MOET programs and under the circumstances of the present study the usefulness of hormonal and metabolic profiles as predictors of the outcome of this biotechnology was limited.     

Factors influencing embryo transfer success in alpacas—A retrospective study

Embryo transfer offers great advantages to South American camelid farmers to reach their breeding goals but the technology still plays a relatively minor role in comparison to other domestic farm animals like cattle. The aim of the present study was to analyse a data set of 5547 single or multiple ovulation embryo transfers performed in commercial alpaca farms in Australia to determine the factors that influence number and quality of embryos produced, embryo transfer success (percentage of crias born) and gestation length following transfer. Logistic binary regression identified the variables day of flushing after mating, embryo diameter, embryo quality, day of transfer after GnRH, and the age of the recipient to have significant impact on the outcome measure embryo transfer success. Transfer of smaller embryos or lower quality embryos resulted in decreased transfer success rates. Optimal days for obtaining embryos from donors were Days 8 and 9 after mating, optimal days for transfer into recipients were Days 7 and 8 after GnRH treatment. Age (>15 years) and body condition of recipients <2 also lowered transfer success rates, while the summer heat had no adverse impact. However, season did influence gestation length, while cria gender did not. In conclusion, results from the analysis of this very large dataset can underpin new recommendations to improve embryo transfer success in alpacas.     

The effect of internal and external factors on bovine embryo transfer results in a tropical environment

The objectives of this work were to determine the effect of external (synchronization methods, month, embryo origin and farm effects) and internal factors (age and size of CL, embryo development and quality score, synchronization methods, age of recipient, quality of transfer and reuse of recipients) on a commercial embryo transfer program in a tropical environment. In the program 1466 Holstein-Friesian purchased embryos were implanted to zebu/European crossbred recipients under field conditions. There were 502 pregnancies detected in this large-scale extension programme. Synchronization methods, month, embryo origin, and farm effects were found to have affected the success rate of embryo transfer. Due to the hot climate and large distances between recipient farms, seasonal effects, reused recipient pregnancy results and the effect of embryo development stage differed from previously reported results. Investigation by ultrasonograph showed that embryo loss occurred before 35 days of pregnancy. Under field conditions, routine fetal sexing resulted in <5% misidentification. In conclusion, under tropical conditions external factors have a major influence on the results of pregnancy from embryo transfer.     

Forty years of embryo transfer in cattle: A review focusing on the journal Theriogenology, the growth of the industry in North America,and personal reminisces

After the first successful transfer of mammalian embryos in 1890, it was approximately 60 years before significant progress was reported in the basic technology of embryo transfer (ET) in cattle. Starting in the early 1970s, technology had progressed sufficiently to support the founding of commercial ET programs in several countries. Today, well-established and reliable techniques involving superovulation, embryo recovery and transfer, cryopreservation, and IVF are utilized worldwide in hundreds, if not thousands, of commercial businesses located in many countries. The mean number of embryos produced via superovulation has changed little in 40 years, but there have been improvements in synchrony and hormonal protocols. Cryopreservation of in vivo-derived embryos is a reliable procedure, but improvements are needed for biopsied and in vitro-derived embryos. High pregnancy rates are achieved when good quality embryos are transferred into suitable recipients and low pregnancy rates are often owing to problems in recipient management and not technology per se. In the future, unanticipated disease outbreaks and the ever-changing economics of cattle and milk prices will continue to influence the ET industry. The issue of abnormal pregnancies involving in vitro embryos has not been satisfactorily resolved and the involvement of abnormal epigenetics associate with this technology merits continued research. Last, genomic testing of bovine embryos is likely to be available in the foreseeable future. This may markedly decrease the number of embryos that are actually transferred and stimulate the evolution of more sophisticated ET businesses.     

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.     

Production and breeding of transgenic cattle using in vitro embryo production technology

Transgenic technology permits major modifications of phenotype by introducing subtle changes in genotype. For domestic farm species, genetic modification may be used to enhance agricultural production or to generate novel genotypes capable of producing heterologous proteins for biomedical applications. The advent of in vitro embryo production techniques has facilitated the large-scale, commercial use of transgenic technology in cattle. Accordingly, we employed in vitro-produced zygotes and embryos in an effort to generate transgenic cattle. Overall, pronuclei in 36,530 in vitro matured and fertilized zygotes were microinjected with a construct designed to express human alpha-lactalbumin in the mammary gland. Of these, 1,472 developed and were transferred to recipients, including 148 twin transfers. Initial pregnancy rate on Day 30 of gestation was 28% (374/1,324). Subsequent calving rate was 17% (226/1,324). Eighteen calves (8%) were transgenic. In vitro produced embryos were used to facilitate breeding of transgenic bulls. Frequency of transgene transmission varied from 3 to 54% between bulls, indicating varying degrees mosaicism. Embryos produced in vitro by these bulls were biopsied and screened for transgenesis prior to transfer to recipients; so far all (6/6) calves born from screened, transgenic embryos were themselves transgenic.     

The current status and future of commercial embryo transfer in cattle

A commercially viable cattle embryo transfer (ET) industry was established in North America during the early 1970s, approximately 80 years after the first successful embryo transfer was reported in a mammal. Initially, techniques for recovering and transferring cattle embryos were exclusively surgical. However, by the late 1970s, most embryos were recovered and transferred nonsurgically. Successful cryopreservation of embryos was widespread by the early 1980s, followed by the introduction of embryo splitting, in vitro procedures, direct transfer of frozen embryos and sexing of embryos. The wide spread adoption of ethylene glycol as a cryoprotectant has simplified the thaw-transfer procedures for frozen embryos. The number of embryos recovered annually has not grown appreciably over the last 10 years in North America and Europe; however, there has been significant growth of commercial ET in South America. Within North America, ET activity has been relatively constant in Holstein cattle, whereas there has been a large ET increase in the Angus breed and a concomitant ET decrease in some other beef breeds. Although a number of new technologies have been adopted within the ET industry in the last decade, the basic procedure of superovulation of donor cattle has undergone little improvement over the last 20 years. The export-import of frozen cattle embryos has become a well-established industry, governed by specific health regulations. The international movement of embryos is subject to sudden and dramatic disturbances, as exemplified by the 2001 outbreak of foot and mouth disease in Great Britain. It is probable that there will be an increased influence of animal rights issues on the ET industry in the future. Several companies in North America are currently commercially producing cloned cattle. The sexing of bovine semen with the use of flow cytometry is extremely accurate and moderate pregnancy rates in heifers have been achieved in field trials, but sexed semen currently is available in only a few countries and on an extremely limited basis. As of yet, all programs involving the production of transgenic cattle are experimental in nature.     

Patency of the cervical canal in cross-bred female Zebu x Brown Swiss selected for non-surgical recovery or transfer of embryos

A test to evaluate the degree of patency of the cervical canal was carried out on 260 Zebu x Brown Swiss cows and heifers that were assigned for non-surgical recovery or transfer of embryos. A total of 204 females (78.5%) were found to have a patent cervix (P <0.05), compared with 56 (21.5%) which had a blockage or obstruction when an attempt was made to pass a catheter through the cervical canal or the "cervical knot" of the purebred and cross-bred Zebu cows. A smaller percentage (12.89%) of non-patent cervices (P <0.05) were detected in multiparas, compared with 25 and 27.5% in primiparas and nullipara, respectively. The test for cervical patency is carried out on Bos indicus cattle to screen the animals before non-surgical embryo transfer manipulations and to attain better results in the application of non-surgical recovery and transfer of embryos.     

Use of assisted reproduction for the improvement of milk production in dairy camels (Camelus dromedarius)

Despite their production potential and ability to survive on marginal resources in extreme conditions, dromedaries have not been exploited as an important food source. Camels have not been specifically selected for milk production, and genetic improvement has been negligible. High individual variation in milk production both within the population and within breeds provides a good base for selection and genetic progress. In this paper, we discuss the possibilities and constraints of selective breeding for milk production in camels, and include a summary of the use of embryo transfer at the world's first camel dairy farm. Embryo transfer is an integral part of the breeding strategy at the camel dairy farm because it increases selection intensity and decreases the generation interval. Using high milk-producing camels as donors and low producing camels as recipients, 146 embryos were recovered (6.1 ± 1.0 embryos/donor; range: 0–18). Embryos were transferred non-surgically into 111 recipients (83 single and 28 twin embryo transfers). Pregnancy rate at 21 days and 5 months was 55% (61/111) and 45% (50/111), respectively. Finally, a total of 46 recipients delivered a live calf. These results document the utility of embryo transfer using high milk producing dromedaries as donors.     

Production of piglets from in vitro-produced embryos following non-surgical transfer

The objective of this study was to enhance procedures for producing piglets derived from in vitro-produced (IVP) pig embryos by non-surgical embryo transfer (ET). The effects of insertion length for the catheter, asynchrony between the age of donor IVP blastocysts and the recipient estrous cycle, and volume of transfer medium were investigated. The IVP blastocysts at 5 days after in vitro fertilization were placed into porcine zygote medium (PZM)-5 supplemented with 10% (v/v) fetal bovine serum (PZM+FBS) in a 0.25 mL plastic straw (21-40 blastocysts per straw) and then transferred into one uterine horn of recipients using the Takumi(?) catheter for deep intrauterine insertion. Successful production of piglets derived from IVP embryos was achieved following non-surgical ET when the catheter was inserted at more than 30 cm anterior to the spiral guide spirette. The efficiency of piglet production (percentage number of piglet(s) born based on the number of embryos transferred) was greater (P<0.05) in recipients whose estrous cycle was asynchronous to that of donors with a 1-day delay (8.3%) than in those with a 2-day (1.5%) or 3-day (0.9%) delay, while pregnancy and farrowing rates (10-40%) did not differ among treatments. When blastocysts were transferred into recipients with 1.0 or 2.5 mL PZM+FBS, there were no significant differences in farrowing rate (30-40%) or average litter size (4.5-6.7) between treatments. The results of the present study indicate that the insertion length of the deep intrauterine catheter and the degree of asynchrony between donor embryos and recipient estrous cycle influenced on pregnancy and birth outcome following non-surgical transfer of IVP blastocysts.     

Embryo transfer in cattle experience of twenty-four completed cases

Thirty-nine potential embryo transfer and 13 ovum collection experiments were initiated within a small experimental herd of mainly nulliparous cattle. Donors for 50 of the experiments were treated with pregnant mares' serum gonadotrophin (PMSG) with or without human chorionic gonadotrophin (HCG) to induce superovulation. Optimal responses (3–20 ovulations) occurred in 56.4 % of treated animals; there was some evidence of reduced responsiveness to repeated treatment. Overall ovum and embryo recovery rate at slaughter or laparotomy was 46.3 % but in some experiments exceeded 80 % on Days 4 and 5 after estrus. Recovery rates were adversely affected by previous surgery and by excessive ovulation rates. 73% of recovered ova were fertilized. Only 20 of the 39 potential transfer experiments were completed, 15 of the 19 incomplete experiments being due to donor failures (lack of superovulation in 11 cases, no fertilization in four). Transfers of three embryos (one case), two embryos (19 cases) or one embryo (four cases) were made surgically to 24 recipients. Of the first 10 recipients, one conceived; of the last 14, ten (71.4%) did so. Best, results were obtained by collecting ova on Day 5 or 6 and using recipients within one day of being synchronous with the donor. Embryos collected at slaughter gave results as good as did those collected surgically. Ten of the pregnant recipients had received more than one embryo but only four gave birth to more than one calf. Some problems associated with the practical application of this technique are discussed.     

Advanced reproductive technology in the water buffalo

Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic. Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine oocytes and subsequent development to the blastocyst stage have been reported.     

Effect of recombinant bovine somatotropin on superovulatory response and recipient pregnancy rates in a commercial embryo transfer program

Recombinant bovine somatotropin (rbST) has been shown to increase follicular growth in cattle and some studies have demonstrated an increase in superovulatory response for rbST-treated cows. Pregnancy rates have also been shown to increase when rbST was administered around the time of insemination or prior to embryo transfer. The application of rbST for the purpose of increasing superovulatory responses of donor cows and increasing pregnancy rates of recipient heifers was tested in a commercial embryo transfer program. In Experiment 1, embryo donor cows (n = 56) underwent three cycles of control superovulation (two before and one after weaning) and subsequently underwent up to four additional superovulations while being treated with either rbST (500 mg sustained-release rbST; Posilac, Monsanto, St. Louis, MO; n = 28) or excipient (control; n = 28) once every 14 days. In Experiment 2, lactating embryo donor cows (n = 37) underwent a control superovulation and then underwent a superovulation while lactating and being treated with either rbST (n = 16) or excipient (n = 21). In Experiment 3, embryo recipient heifers that were being implanted with either in vitro or in vivo produced embryos were treated with either rbST (n = 146) or excipient (n = 143) at the time of embryo transfer. Treatment of non-lactating (Experiment 1) or lactating (Experiment 2) donor cows with rbST during repeated superovulation did not affect the number of corpora lutea, the sum of transferable embryos, degenerate embryos, and unfertilized oocytes, or the number of transferable embryos. Treatment of recipient heifers with rbST (Experiment 3) did not affect pregnancy rates for either in vitro or in vivo produced embryos. We conclude that superovulatory response and pregnancy rates (respectively) are similar to control for rbST-treated cows undergoing repeated superovulations and rbST-treated recipient heifers treated at the time of embryo transfer.     

Collection and transfer of multiple embryos in the mare

A pituitary extract was used to induce multiple ovulations in mares to determine whether day-7 embryos from multiple ovulators were viable as indicated by their ability to develop when transferred to recipients. There were more ovulations/donor for induced multiple-ovulating mares than for control single-ovulating mares (4.6 +/- 0.5 vs 1.0 +/- 0.0; n=14). The embryo collection rate per ovulation was similar for multiple ovulators (0.6 +/- 0.1 embryos/ovulation) and single ovulators (0.7 +/- 0.1). The embryo collection rate per donor, therefore, was higher (P<0.01) for the multiple ovulators (2.9 +/- 0.7 vs 0.7 +/- 0.1). The transfer success rate per embryo at day 21 was different (P<0.05) among recipients which received an embryo from control single-ovulating donors (7 8 ), multiple ovulators from which a single embryo was recovered (2 2 ), and multiple ovulators from which multiple embryos were recovered (9 19 ). The recipient pregnancy rate/donor at day 21 was 88% (7 8 ) for single-ovulating controls and 138% (11 8 ) for induced multiple ovulators. Results indicate that the survivability of day-7 embryos from multiple-ovulating donors was reduced. However, despite the reduced survival rate/embryo, the number of pregnant recipients/donor was increased by induction of multiple ovulations because of the increased number of embryos available for transfer.     

In vitro production of bovine embryos using sex-sorted sperm

The objective of this study was to investigate the suitability of sex-sorted sperm for producing viable in vitro embryos for subsequent transfer into recipient cows and heifers on commercial dairy farms. From August 2002 to June 2003, ovaries were collected from 104 producer-nominated Holstein donor cows on seven Wisconsin farms via colpotomy or at slaughter. Oocytes (N=3526) were aspirated from these ovaries, fertilized 22+/-0.2h later, and cultured to the morula or blastocyst stage. The fluorescence-activated cell sorting ("Beltsville") approach was used to produce (primarily) X-bearing sperm from the ejaculates of three young Holstein sires, and 365 transferable embryos were produced. On average, 3.6+/-0.3 (means+/-S.E.M.) transferable embryos were produced per donor, including 1.4+/-0.2 (Grade 1), 1.5+/-0.2 (Grade 2), and 0.7+/-0.1 (Grade 3) embryos. Number of usable oocytes per donor (33.9+/-3.3) and percent cleavage (51.1+/-1.9) were significant predictors of the number of blastocysts that developed. Mean conception rates for the resulting in vitro embryos were 34.2+/-1.6% in yearling heifer recipients and 18.2+/-0.7% in lactating cow recipients. Additional oocytes (N=3312) from ovaries of anonymous donors (N unknown) collected at a commercial abattoir were fertilized using unsorted sperm, and the percentage of these that developed to blastocyst stage (20.1+/-2.9) was greater (P<0.05) than the corresponding percentage (12.2+/-2.3) achieved with sex-sorted sperm using oocytes (N=1577) from the same source. In summary, we inferred that in vitro embryo production may be a promising application of sex-sorted sperm in dairy cattle breeding, but that the biological causes of impaired embryo development in vitro and compromised conception rates of transferred embryos should be further investigated.     

Influence of environmental temperature on reproductive performance of bovine embryo donors and recipients in the southwest region of the United States

Superovulation and embryo transfer records on performance of embryo donor (n = 3,908; beef 99%, dairy 1%) and recipient (n = 19,936; beef 92%, dairy 8%) cattle from a commercial transfer unit were analyzed for environmental effects. Embryos (n = 42,428) were recovered on Days 5 to 8 postestrus from superovulated donors. Numbers of ova, fertilized ova and embryos of transferable quality were recorded. Transferable embryos were classified according to stage of development and morphological quality. Embryos (n = 19,936) were transferred nonsurgically. Responses were modeled with maximum-likelihood procedures using log-linear functions of independent variables and ANOVA. Fluctuations in daily maximum temperature (1 to 43 degrees C), for Days 0 to 7 of embryo development, had no effect on distribution of embryos classified as good (48%), fair (40%) and poor (12%). Temperature did not affect the percentage of donors flushed with recoverable ova (89%), mean number of ova (12.2 +/- 0.3), fertilization rate (76%) or percentage of transferable embryos (57%). Recipient pregnancy rate (56%) was not affected by mean maximum temperature for Days 0 to 10 posttransfer. Interactions between temperature and breed type (dairy vs beef), parity (cow vs heifer), or lactational status (lactating vs dry) on pregnancy rate were recorded. Elevated environmental temperature does not appear to adversely affect reproductive responses of donor and recipient cattle intensely managed in a commercial transfer unit.     

First successful transfer of cryopreserved feline (Felis catus) embryos resulting in live offspring

Sexually mature domestic cats were hormonally stimulated to induce superovulation; embryo recovery was accomplished by laparotomy. Embryos were frozen by conventional embryo freezing methods used in the domestic cattle embryo transfer industry. Thawing was achieved in a 28 degrees C or 37 degrees C waterbath or in ambient air. Overnight culture of the frozen-thawed embryos in a supplemented Nutrient Mixture F-10 (Ham's) or Earle's Balanced Salt Solution with 20% heat-treated newborn calf serum resulted in five successful term litters from recipient queens. Embryo recipients who became pregnant had been treated with a subcutaneous injection of follicle-stimulating hormone (FSH-P) once every 24 hr for 6 days in the amount of 0.2 mg/day for the first 5 days and 0.1 mg on the sixth day, followed by two intramuscular 750 IU injections of human chorionic gonadotropin 24 hr apart, beginning on the same day as the sixth injection of FSH-P.