Embryo Transfer Technology Research Articles

Scanning and transmission electron microscopic analysis of ampullary segment of oviduct during estrous cycle in caprines.

The ampullary segment of the mammalian oviduct provides suitable milieu for fertilization and development of zygote before implantation into uterus. It is, therefore, in the present study, the cyclic changes in the morphology of ampullary segment of goat oviduct were studied during follicular and luteal phases using scanning and transmission electron microscopy techniques. Topographical analysis revealed the presence of uniformly ciliated ampullary epithelia, concealing apical processes of non-ciliated cells along with bulbous secretory cells during follicular phase. The luteal phase was marked with decline in number of ciliated cells with increased occurrence of secretory cells. The ultrastructure analysis has demonstrated the presence of indented nuclear membrane, supranuclear cytoplasm, secretory granules, rough endoplasmic reticulum, large lipid droplets, apically located glycogen masses, oval shaped mitochondria in the secretory cells. The ciliated cells were characterized by the presence of elongated nuclei, abundant smooth endoplasmic reticulum, oval or spherical shaped mitochondria with crecentric cristae during follicular phase. However, in the luteal phase, secretory cells were possessing highly indented nucleus with diffused electron dense chromatin, hyaline nucleosol, increased number of lipid droplets. The ciliated cells had numerous fibrous granules and basal bodies. The parallel use of scanning and transmission electron microscopy techniques has enabled us to examine the cyclic and hormone dependent changes occurring in the topography and fine structure of epithelium of ampullary segment and its cells during different reproductive phases that will be great help in understanding major bottle neck that limits success rate in vitro fertilization and embryo transfer technology.

Preliminary study of factors affecting the superovulatory response of high producing dairy cows superstimulated regardless of the stage of estrous cycle in Egypt

This work was conducted as a first time commercial production of embryos from lactating Holstein and Brown Swiss cows using multiple ovulation embryo transfer (MOET) technology in Egypt. We studied factors affecting the superovulatory response (SR) in superovulated cows and effects of propylene glycol (PG) on embryo quality. Daily milk production at flushing had significantly negative effects on SR and embryo yields in superovulated cows. In addition, Brown Swiss cows had better SR than Holstein cows. Moreover, cows having more than 3 parities yielded better response, compared to cows in the first three parities. However, factors such as body weight at flushing, body condition score (BCS) at flushing and days in milk (DIM) at flushing did not have any association with SR in cows. In addition, drenching of PG prior to and during the superovulatory treatment improved SR (Right CL number, P < 0.05; Left CL number, P < 0.05), total embryos per flush (P < 0.05), first grade embryos per flush (P < 0.01) and tended to improve transferable embryos (P = 0.13) and second grade embryos per flush (P = 0.11). However, it tended to increase the number of degenerated embryos per flush (0.06). In conclusion, commercial production of embryos from lactating Holstein and Brown Swiss cows regardless of stage of estrous cycle by MOET proved successful under Egyptian conditions. Milk yield was negatively associated with SR and embryo yields in superovulated cows. Moreover, drenching of PG prior to and during the superovulatory treatment improved SR and embryo yields.

Embryo Transfer Technology in Camels: Education Article

Attempts have been made in this manuscript to point out the important aspects of physiology of reproduction of camel which has a major impact on outcome of the technology of embryo transplantation applied in this species.

Production of Middle White Piglets after Transfer of Embryos Produced &lt;i&gt;In Vitro&lt;/i&gt;

The present study was conducted to examine the feasibility of in vitro embryo production and transfer technologies for producing Middle White piglets. After collection from three retired Middle White sows, a total of 222 oocytes were matured, fertilized and cultured in vitro, and a total of 50 embryos from the 4-cell to blastocyst stage were produced by the 4th or 5th day. These embryos were transferred individually into three recipients along with 5 in vivo-derived Duroc blastocysts. All of the recipients became pregnant, and they farrowed a total of 9 Middle White and 9 Duroc piglets. These results suggest that in vitro embryo production using ovaries from retired sows is useful for reproduction of pigs of pure breeds including the Middle White for breeding activities and conservation/utilization of genetic resources.

수정란 이식 기술의 응용

수정란 이식 기술의 응용

Integrating genomic selection into dairy cattle breeding programmes: a review

Extensive genetic progress has been achieved in dairy cattle populations on many traits of economic importance because of efficient breeding programmes. Success of these programmes has relied on progeny testing of the best young males to accurately assess their genetic merit and hence their potential for breeding. Over the last few years, the integration of dense genomic information into statistical tools used to make selection decisions, commonly referred to as genomic selection, has enabled gains in predicting accuracy of breeding values for young animals without own performance. The possibility to select animals at an early stage allows defining new breeding strategies aimed at boosting genetic progress while reducing costs. The first objective of this article was to review methods used to model and optimize breeding schemes integrating genomic selection and to discuss their relative advantages and limitations. The second objective was to summarize the main results and perspectives on the use of genomic selection in practical breeding schemes, on the basis of the example of dairy cattle populations. Two main designs of breeding programmes integrating genomic selection were studied in dairy cattle. Genomic selection can be used either for pre-selecting males to be progeny tested or for selecting males to be used as active sires in the population. The first option produces moderate genetic gains without changing the structure of breeding programmes. The second option leads to large genetic gains, up to double those of conventional schemes because of a major reduction in the mean generation interval, but it requires greater changes in breeding programme structure. The literature suggests that genomic selection becomes more attractive when it is coupled with embryo transfer technologies to further increase selection intensity on the dam-to-sire pathway. The use of genomic information also offers new opportunities to improve preservation of genetic variation. However, recent simulation studies have shown that putting constraints on genomic inbreeding rates for defining optimal contributions of breeding animals could significantly reduce achievable genetic gain. Finally, the article summarizes the potential of genomic selection to include new traits in the breeding goal to meet societal demands regarding animal health and environmental efficiency in animal production.

Cryopreservation of Canine Embryos and Resulting Pregnancies

The assisted reproductive techniques used in dogs have strictly limited utility when compared with other mammals. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen has been only readily available method for improved breeding for companion and working dogs. Recently, successful cryopreservation of embryos and subsequent embryo transfer with a non-surgical technique in dog was reported. Application of embryo cryopreservation and transfer technology could contribute to breeding management in companion dogs, working dogs including guide dogs and drug-detecting dogs and quarantine dogs. Such technology would also facilitate the transportation and storage of genetic materials and aid in the elimination of vertically transmitting diseases in the dog.

Current Status and the Future of Bovine Embryo Transfer using Sex Determined Embryos

The embryo transfer technology represents a powerful tool for the acceleration of various breeding programs in cattle. In cattle breeding, it is important to know the sex of embryos in order to improve the genetic potential of the herd. Normally, only five to six calves can be obtained during the reproductive life-time of a cow. However, calving rates can be increased by superovulation and embryo transfer program. If embryo sexing techniques are applied for the program, embryos with the preferred sex can be obtained from cows with high genetic performance. This is a potent way to improve the genetic background and production of animals. However, the reliability, cost, practicality and time required for this approach must be considered as well. The present paper discusses the current status and the future of bovine embryo transfer using sex determined embryos.

Treatments for the synchronisation of bovine recipients for fixed-time embryo transfer and improvement of pregnancy rates

Although embryo transfer technology has been used commercially in cattle for many years, the inefficiency of oestrus detection, especially in recipients, has limited the widespread application of this technology. The most useful alternative to increase the number of recipients utilised in an embryo transfer program is the use of protocols that allow for embryo transfer without the need for oestrus detection, usually called fixed-time embryo transfer (FTET). Most current FTET protocols are based on progestin-releasing devices combined with oestradiol or GnRH, which control and synchronise follicular wave dynamics and ovulation. Conception rates to a single FTET have been reported to be similar to those after detection of oestrus, but pregnancy rates are higher because these treatments have increased the proportion of recipients that receive an embryo. Recent changes to treatments for FTET, such as the administration of eCG, have resulted in increased pregnancy rates and provide opportunities to make these treatments easier to perform on farm.

Perspectives for feed-efficient animal production1

Modern animal breeding programs are largely based on biotechnological procedures, including AI and embryo transfer technology. Recent breakthroughs in reproductive technologies, such as somatic cell nuclear transfer and in vitro embryo production, and their combination with the emerging molecular genetic tools, will further advance progress and provide new opportunities for livestock breeding. This is urgently needed in light of the global challenges such as the ever-increasing human population, the limited resources of arable land, and the urgent environmental problems associated with farm animal production. Here, we focus on genomic breeding strategies and transgenic approaches for making farm animals more feed efficient. Based on studies in the mouse and rat model, we have identified a panel of genes that are critically involved in the regulation of feed uptake and that could contribute toward future breeding of farm animals with reduced environmental impact. We anticipate that genetically modified animals will play a significant role in shaping the future of feed-efficient and thus sustainable animal production, but will develop more slowly than the biomedical applications because of the complexity of the regulation of feed intake and metabolism.

Animal breeding in India - a time for reflection, and action

Animal breeding in India - a time for reflection, and action

Embryo transfer: a comparative biosecurity avantage in international movements of germplasm

This paper uses cattle as a model to provide an overview of the hazards involved in the transfer of in vivo-derived and in vitro-produced embryos. While scientific studies in recent decades have led to the identification of pathogens that may be associated with both in vivo- and in vitro-derived embryos, those studies have also been the basis of appropriate disease control measures to reduce the risks to a negligible level. These disease control measures have been identified and assessed by the International Embryo Transfer Society's (lETS) Health and Safety Advisory Committee, the expert body that advises the World Organisation for Animal Health (OIE) on matters related to the safety of embryo transfer. Through the OIE's processes for developing and adopting international standards, the disease control measures identified by the IETS have been incorporated into the Terrestrial Animal Health Code. The basic principles rely on the crucial ethical roles of the embryo collection team and embryo transfer team, under the leadership of approved veterinarians. Decades of experience, with nearly 10 million embryos transferred, have demonstrated the very significant biosecurity advantage that embryo transfer technology has when moving germplasm internationally, provided that the international standards developed by the IETS and adopted by the OIE are strictly followed.

Scanning electron microscopic changes in granulosa cells during follicular atresia in Caprine ovary

During this study, topographic changes in healthy and atretic granulosa cells have been investigated during follicular atresia in goat ovary. Under scanning electron microscopy atresia was marked by asymmetrical shrinkage and vacuolization of cytoplasm. The specific topographical alterations observed in atretic cells were loss of micro extensions, disruption of cell-cell interaction, and smooth-textured membrane with a number of uneven depressions and ruffles. Some portions of the cell membrane were marked by extensive shrinkage due to condensation of cytosol. Irregular membrane at occasions was studded with blunt microextensions. The findings of present investigation will help in understanding the cellular changes in granulosa cells during follicular atresia and will find applications in screening of follicles for in vitro culture, in vitro fertilization and Embryo transfer technology.

Oestrous Synchronization, Ovarian Superovulation and Intraspecific Transfers From a Closed Breeding Colony of Inbred SLA Miniature Pigs

The inbred SLA miniature pig is a unique animal model developed for organ transplantation studies and pre-clinical experimental purposes. Reported oestrous synchronization and superovulation treatments were examined in two SLA haplotypes (AA and DD) to allow collection of embryos for both practical embryo transfer and experimental technologies from a closed breeding colony. Pre-puberal miniature pigs were poor responders to oestrous synchronization treatments, while post-puberal sows were equivalent to commercial sows. Following superovulation, the ovulation number (corpora .hemorrhagica) was higher (p < 0.05) in the cycling sows when compared with non-cycling sows. Ovulations were equivalent to commercial pre-puberal gilts and non-cycling sows (p > 0.05). No difference in ovulation number between haplotypes was observed, which differs from the previous report (DD>AA). Collection of zygotes for pronuclear injection was the highest in the non-cycling post-puberal miniature pig group (p < 0.05), although significantly lower when compared with the commercial pig treatment groups (p < 0.05). The incidence of cystic endometrial hyperplasia in our colony was equivalent to rates observed in commercial pigs. Pronuclear visualization following centrifugation was the highest in the non-cycling miniature sow group and approximates to about 25% of ovulations and about half the rate observed in the commercial pigs (50%). Miniature pig embryos transferred between SLA haplotypes and transfer of DD embryos to commercial pigs resulted in live births at a higher efficiency than previously reported. This study demonstrates the feasibility of undertaking assisted reproductive technologies in a closed breeding colony of inbred SLA miniature pigs without compromise to the breeding programmes.

Changes in Trace Elements during Follicular Atresia in Goat (Capra hircus) Ovary

Quantitative analysis of follicular fluid and granulosa cells from small, medium and large antral atretic follicles of goat (Capra hircus) ovaries was conducted to study the alterations in trace elements viz zinc (Zn), copper (Cu), manganese (Mn), and iron (Fe). The zinc content was lower in the follicular fluid (0.993 ± 0.001, 0.935 ± 0.002, 1.321 ± 0.001 μg/ml) and granulosa cells (0.867 ± 0.002, 0.801 ± 0.001, 1.073 ± 0.002 μg/mg) of small, medium, and large antral atretic follicles respectively than their respective controls. Copper quantity was higher in the follicular fluid (0.113 ± 0.001, [Formula: see text], 0.224 ± 0.001 μg/ml) and granulosa cells (0.094 ± 0.001, 0.114 ± 0.001, 0.182 ± 0.001 μg/mg) from small, medium, and large antral atretic follicles respectively than their respective controls. Similarly, iron and manganese was also found higher in the follicular fluid and granulosa cells of small, medium, and large antral atretic follicles than their respective controls. The present study provides the basic data on trace elements that can be safely used as atretic marker and will find use in in vitro studies for fertility improvement plan. Thus, help in elevating the number of ovulations and screening of follicles to enhance the success rate in vivo and in vitro fertilization and embryo transfer technology.

240 DIFFERENCES IN GLOBAL TRANSCRIPTOME PROFILE OF BOVINE BLASTOCYSTS DERIVED FROM SUPEROVULATED OR SYNCHRONIZED CYCLIC HEIFERS

Estrus synchronization and superovulation are the most widely used procedures in embryo transfer technology. However, changes in the oviduct and uterine environment due to these procedures and the subsequent influence on embryos have not yet been investigated. This study was con- ducted to investigate the effect of oviduct environment of only synchronized or superovulated cyclic heifers on the gene expression profile of blastocysts. Bovine Affymetrix array analysis was performed using 2 groups of blastocysts. The first group was bovine blastocysts produced after superovulation of Simmental heifers (n = 9) using 8 consecutive FSH injections over 4 days in decreasing doses (in total, 300-400 mg of FSH equivalent according to body weight) and flushed at Day 7 by nonsurgical endoscopic method. The second group was bovine blastocysts derived from synchronized Simmental heifers (n = 4) after transfer of 2-cell stage embryos from superovulated donor Simmental heifers (n = 9) by nonsurgical transvaginal endoscopy tubal transfer method. Total RNA was extracted from 3 pools of embryos from each experimental group (6 embryos per pool). A total of 6 biotin-labeled cRNA samples were hybridized on 6 bovine Affymetrix arrays. Data analysis was performed using LIMMA written on R package, which maintained the Bioconductor. Array data analysis revealed a total of 454 transcripts to be differen- tially expressed (P &lt; 0.05, fold change &gt;2) between the 2 groups. Of these, 429 and 25 were up- and down-regulated, respectively, in blastocysts derived from superovulated heifers compared with those derived from synchronized animals. Genes involved in response to stress (HSPA14 and HSPE1), cellular and metabolic processes (CPSF3, ATPIF1, POMP, and MDH2), translation (RPS17, EEF1B2, and EIF4E), and cell commu- nication (FN1, KRT18, and DSG2) were found to be enriched in blastocysts derived from superovulated animals. On the other hand, protein metabolic processes related genes (CLGN) were found to be enriched in blastocysts derived from the synchronized group. The KEGG analysis of the differentially expressed genes showed that the ribosome and oxidative phosphorylation pathways are the dominant pathways and genes involved in these pathways are greatly abundant in the blastocysts derived from superovulated animals. Quantitative real-time PCR has confirmed the transcript abundance of 7 out of 8 genes selected for validation. In conclusion, blastocysts cultured in synchronized animals post 2-cell stage showed significant differences in transcriptome profile compared with their counterparts that remained in superovulated heifers until Day 7. Further functional analysis of some selected candidate genes could give new insights into mechanisms regulating the ability of embryos to survive after transfer.

New approaches to superovulation and embryo transfer in small ruminants

The present paper reviews the current state of knowledge in multiple ovulation and embryo transfer (MOET) technology in small ruminants, focusing on recently reported information. Major new findings are related to follicular wave patterns in small ruminants, the elucidation of follicular dominance and the integration of this information into ovarian superstimulatory treatment protocols. Follicular dynamics determine steroid and gonadotrophin secretion, follicular responses to FSH, ovulatory responses and embryo yield. Protocols that control follicular dominance have been designed to allow the initiation of superstimulation at the beginning of a follicular wave. New approaches consist of (1) synchronisation of ovulation for superstimulation during Wave 1 (i.e. Day 0 protocol), (2) pretreatment with a gonadotrophin-releasing hormone (GnRH) antagonist from 10 days prior to FSH treatment to avoid follicular dominance and (3) progesterone-oestradiol cotreatment to synchronise follicle wave emergence. These protocols provide a homogeneous pool of small follicles that are gonadotrophin responsive, enhancing the superovulatory response and embryo yield with a reduction in the incidence of unovulated follicles and early regression of corpora lutea. In addition, the rate of fertilisation failure has been reduced by using an inducer of ovulation (i.e. GnRH) associated with intrauterine insemination. In summary, the application of recently acquired knowledge has resulted in relevant improvements in MOET programmes in small ruminants.

New approaches to superovulation in the cow

There is continuing need to simplify bovine superovulation protocols without compromising embryo production. The control of follicular wave emergence and ovulation has facilitated donor management, but the most commonly used treatment, oestradiol, cannot be used in many parts of the world and mechanical removal of the dominant follicle is difficult to apply in the field. Other alternatives include gonadotrophin-releasing hormone (GnRH) or LH, but efficacy in groups of randomly cycling animals is variable. Another alternative is to increase the response to GnRH by inducing a persistent follicle and initiating FSH treatments following GnRH-induced ovulation. The number of transferable embryos following superovulation during the first follicular wave did not differ from that achieved 4 days after oestradiol benzoate and progesterone. To further simplify superovulation, FSH has been administered as a single intramuscular injection. Superovulation of beef donors with a single intramuscular injection of Folltropin-V (Bioniche Animal Health, Belleville, ON, Canada) diluted in a slow-release formulation resulted in embryo production comparable to that obtained using the traditional twice-daily protocol. The single intramuscular injection has the potential to reduce labour and handling and may be useful when handling stress is an impediment to success. These alternatives provide ways of facilitating widespread application of embryo transfer technologies.

Simulation study on the efficiencies of MOET nucleus breeding schemes applying marker assisted selection in dairy cattle

Advantages of breeding schemes using genetic marker information and/or multiple ovulation and embryo transfer (MOET) technology over the traditional approach were extensively evaluated through simulation. Milk yield was the trait of interest and QTL was the genetic marker utilized. Eight dairy cattle breeding scenarios were considered, i.e., traditional progeny testing breeding scheme (denoted as STANPT), GASPT scheme including a pre-selection of young bulls entering progeny testing based on their own QTL information, MOETPT scheme using MOET technology to generate young bulls and a selection of young bulls limited within the full-sib family, GAMOPT scheme adopting both QTL pre-selection and MOET technology, COMBPT scheme using a mixed linear model which considered QTL genotype instead of the BLUP model in GAMOPT, and three non-progeny testing schemes, i.e. the MOET, GAMO and COMB schemes, corresponding to MOETPT, GAMOPT and COMBPT with progeny testing being part of the system. Animals were selected based on their breeding value which was estimated under an animal model framework. Sequential selection over 17 years was performed in the simulations and 30 replicates were designed for each scenario. The influences of using QTL information and MOET technology on favorable QTL allele frequency, true breeding values, polygenetic breeding values and the accumulated genetic superiority were extensively evaluated, for five different populations including active sires, lactating cows, bull dams, bull sires, and young bulls. The results showed that the combined schemes significantly outperformed other approaches wherein accumulated true breeding value progressed. The difference between schemes exclusively using QTL information or MOET technology was not significant. The STANPT scheme was the least efficient among the 8 schemes. The schemes using MOET technology had a higher polygenetic response than others in the 17th year. The increases of frequency of the favorable QTL allele varied more greatly across the 3 male groups than in the lactating cows group. The accumulated genetic superiorities of the GASPT scheme, MOETPT scheme, GAMOPT scheme, COMBPT scheme, MOET scheme, GAMO scheme and COMB scheme over the STANPT scheme were 8.42%, 3.59%, 14.58%, 18.54%, 4.12%, 14.12%, 16.50% in active sires and 2.70%, 5.00%, 11.05%, 12.78%, 7.51%, 17.12%, 25.38% in lactating cows.

Efficient reproduction of cynomolgus monkey using pronuclear embryo transfer technique.

One of the technical bottlenecks in producing nonhuman primate models is that current assisted reproductive techniques, such as in vitro culture and frozen conservation of multicell-stage embryos, often result in poor embryo quality and subsequently lead to low birth rates. We investigated whether pronuclear embryo transfer can be used as an effective means for improving pregnancy and live birth rates of nonhuman primates. We collected 174 metaphase II oocytes by laparoscopy from 22 superovulated mature females and then fertilized these eggs using either in vitro fertilization or intracytoplasmic sperm injection, resulting in a 33.3% and a 50% fertilization rate, respectively. These 66 fertilized pronuclear-stage embryos were then tubally transferred to 30 recipients and led to 7 births and 1 abortion. Importantly, we observed that the highest live birth rate of approximately 64% was obtained when the transfer of pronuclear embryos was performed in the presence of new corpus luteum in the ovary of recipients between 24 h and 36 h after estradiol peak. Therefore, our experiments demonstrate that by matching the critical time window in the recipient's reproductive cycle for achieving optimal embryo-uterine synchrony, pronuclear embryo transfer technology can significantly improve the pregnancy rate and live birth of healthy baby monkeys. This efficient method should be valuable to the systematic efforts in construction of various transgenic primate disease models.