Embryo Transfer Technology Research Articles

Prospects for increasing the utilization of cattle embryo transfer by small-scale milk and meat producers in tropical regions.

Small community cattle farmers in the tropics are facing challenges to deliver quality products whilst under pressure to increase milk and beef yields per cow. These challenges could be partially met by crossbreeding Bos taurus with Bos indicus (F1) cattle utilizing embryo transfer (ET) technology. The Bos taurus infusion would increase milk production, whilst the Bos indicus influence can improve resistance to the harsh environment of the tropics. Here, individuals from existing herds can be used to produce F1 embryos which benefit from hybrid vigour. Resultant female offspring would in turn receive an F1 embryo on reaching breeding maturity. This approach would help to provide a cost-effective, systematic approach to improve productivity in dairy and beef cattle in the tropics.However, full usage of ET, including in vitro applications, in the tropics will require improvements in procedures, resources and education.

GnRH or estradiol benzoate combination with CIDR improves in-vivo embryo production in bovines (Bos indicus and Bos taurus) under subtropics.

Multiple Ovulation and Embryo Transfer (MOET) technology is a potential technique to upgrade livestock species’ genetics. The varied response to super-stimulatory treatments remains one of the limiting factors to this technology’s widespread use. The present study was aimed to improve the superovulation response and in-vivo embryo production by using controlled internal drug release (CIDR)-GnRH or CIDR-EB (Estradiol Benzoate) along with conventional superovulation protocol in Holstein Frisian (HF): Bos taurus; n = 42) and Crossbred (XB: Cholistani (Bos indicus) × HF; n = 28) cows. In the CIDR-GnRH/CIDR-EB treatment, CIDR was implanted in the cows after confirming the presence of a corpus luteum (CL) on the 8th day after estrus. 2 ml GnRH (Lecirelin acetate 0.0262 mg/ml) or 2 mg EB was also administered in CIDR-GnRH/CIDR-EB groups, respectively. Both groups were given super-stimulatory treatment from the 11th day after estrus (FSH in tapering doses twice a day for four consecutive days). On day 13, two doses of 2 ml prostaglandin (75 µg/ml of dextrorotatory cloprostenol) were administered (am: pm), and CIDR was removed the following day. Two artificial inseminations (AI) of the cows were performed (12 h apart) on the 15th day. No CIDR and GnRH/E.B were given in the control group, but the remaining superovulation protocol was the same. Later on, seven days after the first AI, non-surgical embryo flushing was done. The transferable embryos produced from three different superovulation protocols were then transferred into the recipient cows (n = 90) for determining their fertility. Statistical analysis revealed that the number of super-estrus follicles (SEF), multiple corpora lutea (MCL), ovulation/fertilization percentage, fertilized structures recovered (FSR), and transferable embryos (TEs) remained significantly higher (p < 0.05), and days taken for return to estrus (RTE) after embryo collection remained significantly lower (p < 0.05) in CIDR-GnRH (n = 18) and CIDR-EB (n = 15) groups as compared to the control (n = 37). The comparison between XB and HF cows revealed that the TEs production in CIDR-GnRH (XB = 5 vs HF = 13) and CIDR-EB (XB = 6 vs HF = 9) based superovulation protocols were 11.60 ± 4.08 vs 04.31 ± 0.98 and 09.33 ± 1.78 vs 05.22 ± 1.36, respectively. TEs production in XB cows (n = 5) of the CIDR-GnRH group was significantly higher (11.60 ± 4.08) than other groups. On the other hand, the days taken for RTE after embryo collection remained significantly lower (p < 0.05) in HF cows of treatment groups. However, the fertility of TEs was neither affected significantly (p > 0.05) by the superovulation protocol used nor by breed differences among donor cows. In conclusion, using CIDR-GnRH or CIDR-EB along with conventional superovulation protocol may enhance the efficiency of MOET programs in cattle. Furthermore, XB donor cows demonstrated a better performance than HF donor cows under subtropical conditions.

Anti Mullerian Hormone: A Putative Endocrine Marker for Prediction of Superovulation Response in Cattle

Anti-Müllerian Hormone (AMH) is a dimeric glycoprotein molecule with a molecular weight of 140-kDa linked to the Transforming Growth Factor-β (TGF-β) superfamily. Research on the use of AMH in livestock has gained momentum in recent years. In particular, it is now widely used in cattle breeding, where embryo transfer technology is used to obtain more offspring from genetically superior females. One of the most important factors that increase the success of embryo transfer is the response of the selected donor to the superovulation protocol. AMH has been successfully used as a biomarker in predicting superovulation response in cattle and in estimating the numbers of oocytes collected by ovum pick up (OPU). AMH plasma concentrations are positively and highly correlated with antral follicle count (AFC) in cattle and can also be used as a marker of ovarian reserve. In addition, AMH was also positively and highly correlated with the number of corpus luteum (CL) and total embryos after superovulation in several studies. It has been also reported via Genome-Wide Association Studies (GWAS) that plasma AMH level is an inherited trait in cattle and can be improved through genomic selection. In this study, we aimed to evaluate the relationship between plasma AMH levels and superovulation response in cattle by compiling the data obtained from various studies in light of current scientific literature.

МЕДИЧНІ АСПЕКТИ СУРОГАТНОГО МАТЕРИНСТВА

Surrogacy is associated with a wide range of factors that increase maternal and perinatal risks. The use of in vitro fertilization and embryo transfer technologies determines the higher frequency of multiple pregnancies that enhances the risk of cesarean section, premature birth, low body weight in newborns, etc. in case of surrogacy. Genetic differences and immunogenetic reactions increase the risk of pre-eclampsia, placental pathology, abortion, intrauterine growth retardation. Hormonal disbalance under the effect of adverse factors during surrogate pregnancy may initiate prenatal reprogramming of morphogenesis through epigenetic mechanisms. This can affect the organogenesis and predispose to susceptibility to various diseases. Psychological factors and emotional link between a surrogate mother and a fetus are associated with increased levels of hormones of stress-releasing systems, which affect the formation of neurohumoral systems of the fetus and can modulate its mental development.

Patients Who Failed to Conceive Following an In Vitro Fertilization Cycle Can Be Clustered Into Different Failure Causes Using Gene Expression Hierarchical Analysis

(Abstracted from Biol Reprod 2020;103:599–607) The efficacy of in vitro fertilization (IVF) with embryo transfer and other assisted reproductive technologies has dramatically improved since their introduction, resulting in current multiple pregnancy rates lower than 10%. Despite advancements, an overall success rate of 30% could be improved by further exploration of why treatment response remains highly variable between patients.

Curtailing the conventional dose of Folltropin-V for superstimulation and embryo recovery in Sahiwal cattle (Bos indicus)

High cost of the follicle stimulating hormone (FSH) impacted embryo transfer technology (ETT) work in India. The experiment evaluated effects of reduced (200 mg vs 400 mg) FSH (Folltropin-V) dose on superovulation and embryo recovery in Sahiwal cattle. Animals treated with conventional dose of Folltropin, i.e. 400 mg (G1, n=6) were compared with another group administered with half dose (i.e. 200 mg) of Folltropin (G2, n=6). Superstimulatory response (number of follicles &gt;8 mm) during estrus; unovulatory follicles, superovulatory response (one day prior to embryo flushing) using trans-rectal ultrasonography were recorded. Embryos were collected nonsurgically on day-7 post insemination and total recovered and transferable embryos were noted. Results revealed that number of follicles during estrus and subsequent ovulations were significantly higher in the G1 compared to G2 (30.0±1.37 and 26.3±0.99 vs. 20.3±0.21 and 17.7±0.42, respectively). Unovulatory follicles per animal were lower (2.6±0.21 vs. 3.7±0.49) in G2 than G1. The group receiving lower dose of Folltropin (G2) had significantly higher average embryo recovery compared to G1 (12.0±0.63 vs 8.7±0.76, respectively). Additionally, average number of transferable embryos were non-significantly higher in G2 (7.0±0.37) than G1 (5.7±0.88). The study indicated that lower dose of Folltropin-V (200 mg compared to 400 mg) is more effective in superovulation and embryo production in Sahiwal cows.

Research on the Application of Artificial Intelligence Technology in Animal Embryo Transfer

Animal embryo transfer technology can show strong practical value, and its use in the animal husbandry industry is particularly prominent. It should be because this technology can significantly increase the birth rate and survival rate of animal pups within a period of time. This technology can also the animal’s own reproductive ability has been further improved. With the popularization of this animal embryo transfer technology, the animal husbandry industry can achieve rapid development in a short period of time, enabling farmers to obtain higher and better economic benefits. Since animal embryo transfer technology entered the field of animal husbandry in the 1970s, the role of animal embryo transfer technology has become increasingly apparent. This technology has penetrated into biological reproduction technologies such as animal breeding, gender control and animal cloning. And animal reproduction technology has become leading the cutting-edge scientific reproduction technology of the livestock industry in the 21st century. Nowadays, artificial intelligence technology combines the research results of many disciplines such as theoretical science, technical science, and applied science. As a practical technology leading a new generation of technological revolution and industrial reform, artificial intelligence technology has a significant impact on human social progress, economic development, and biotechnology. However, the transformation of artificial intelligence technology has also brought some huge challenges to social stability. How to treat artificial intelligence and animal embryo transfer technology, how to integrate and coordinate these two technologies, and how to solve these technical design problems have become the urgent problems that need to be solved.

Intrauterine Infusion of TGF-β1 Prior to Insemination, Alike Seminal Plasma, Influences Endometrial Cytokine Responses but Does Not Impact the Timing of the Progression of Pre-Implantation Pig Embryo Development.

Simple SummaryAlthough endometrial immune regulation in pigs during the early preimplantation period is poorly documented, particularly under conditions of embryo transfer (ET), it is recognized that seminal plasma (SP) induces molecular changes in the reproductive tract, influencing numerous reproductive functions. A principal constituent of SP is the cytokine transforming growth factor β1 (TGF-β1), which has an important role in embryo development, pregnancy establishment, and progression. The present study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, porcine TGF-β1 in an extender, or an extender alone (control)) by mimicking an ET scenario in so-called “donor” (inseminated) and “recipient” (uninseminated) sows. We investigated the effects of these treatments on day 6 embryo development (“donors”) and endometrial explants’ cytokine production (“donors” and “recipients”). SP infusion positively influenced embryo development compared with TGF-β1 or extender infusions. Infusion treatments differentially affected endometrial cytokine production, with the effects being stronger in “donors” than in “recipients.” Increased knowledge of the effects of SP or some of its active components on the female immune system may help to develop strategies for increasing the reproductive efficiency for the benefit of pig ET.Seminal plasma (SP) in the female genital tract induces changes that affect multiple reproductive processes. One of the active components in SP is the transforming growth factor β1 (TGF-β1), which has major roles in embryo development and pregnancy. Embryo transfer (ET) technology is welcomed by the pig industry provided that embryo quality at embryo collection as well as the fertility and prolificacy of the recipients after the ET is increased. This study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, TGF-β1 cytokine in the extender, or the extender alone (control)) by mimicking an ET scenario in so-called “donor” (inseminated) and “recipient” (uninseminated) sows. On day 6 (day 0—onset of estrus), all “donors” were laparotomized to determine their pregnancy status (presence and developmental stage of the embryos). In addition, endometrial explants were collected from pregnant “donors” and cyclic “recipients,” incubated for 24 h, and analyzed for cytokine production. SP infusions (unlike TGF-β1 infusions) positively influenced the developmental stage of day 6 embryos. Infusion treatments differentially influenced the endometrial cytokine production, mainly in donors. We concluded that SP infusions prior to AI not only impacted the porcine preimplantation embryo development but also influenced the endometrial cytokine production six days after treatment, both in donors and recipients.

76 Effect of season on the superstimulation response, embryo quality, and pregnancy establishment in dromedary camel (Camelus dromedarius)

The present study was conducted to study the effect of season on the superstimulation response, embryo quality and pregnancy establishment in the dromedary camels. Two donors each were superstimulated in the month of June 2019 (non-breeding season) and January 2019 (breeding season) by a combination of 2500IU of equine chorionic gonadotrophin (eCG, Folligon; Intervet), given as a single intramuscular injection on Day 1 of the treatment protocol, and 400mg of pFSH (Folltropin; Bioniche) injected twice daily in declining doses of 2×80mg, 2×60mg, 2×40mg, and 2×20mg over 4 days, also beginning on Day 1. They were scanned by rectal ultrasonography and follicles counted before mating with a fertile bull on Day 11 after the start of treatment. An injection of 20µg of gonadotrophhin-releasing hormone (Receptal, Intervet) was given immediately after mating to induce ovulation. The number of corpora lutea (CL) were counted, and the embryos collected by the non-surgical method on Day 7 after ovulation. All embryos were washed and graded before they were transferred individually into the left uterine horn of recipient animals, synchronized to be on Day 6 after ovulation. All recipients were screened by ultrasonography on Day 22, Day 60, and Day 90 for pregnancies. The data were analysed using a two-sample t-test (Minitab statistical software, Minitab Ltd.). No difference was observed in the mean number of follicles developed (20.5 vs. 18.5) between the donors in breeding and non-breeding season, respectively. The percent mean±s.e.m. of ovulations as observed by counting the CL in nonbreeding season (80.7±3.4) were not different from those in breeding season (85.4±3.7). A total number of 16 embryos collected during non-breeding season and 20 embryos in breeding season were transferred to recipients. The percent mean±s.e.m. of pregnancies established on Day 22 (56.6±3.4 vs. 67.5±7.5) and on Day 90 (47.2±2.8 vs. 60±0.0) were similar in both groups. These observations indicate that embryo transfer technology can be applied to camels during the summer months, which is considered the non-breeding season, and pregnancies can be established at par with breeding season. In conclusion, to the best of our knowledge, this is the first study in camels wherein we have demonstrated that donor camels can be superstimulated, embryos can be collected, recipient animals can be synchronized, and pregnancies can be established in the summer (non-breeding) months. The embryos collected from elite donors can be transferred to synchronized recipients to establish pregnancies or preserved for future use. Some owners do not want calves to be born in summer months, so protocols need to be established for storing these embryos for a few months before their transfer to recipients, to have the calves delivered in the desired winter months.

73 Use of embryo transfer technology to salvage the germplasm of elite dromedary camels (Camelus dromedarius) infected with brucellosis

The present study was conducted to evaluate the potential of embryo transfer technology to salvage invivo-produced embryos from elite dromedary camels infected with brucellosis without transmission of the pathogen to the recipients or offspring. Ten elite racing champions who tested positive for brucella comprised the experimental group, whereas 3 donors negative for brucella acted as the control. Each donor animal received a combination of 2500IU of equine chorionic gonadotrophin (eCG; Folligon; Intervet), given as a single intramuscular injection on Day 1 of the treatment protocol, and 400mg of pFSH (Folltropin; Bioniche), injected twice daily in declining doses of 2×80mg, 2×60mg, 2×40mg, and 2×20mg over 4 days, also beginning on Day 1. They were mated with a fertile bull on the 11th day after the start of treatment. The donors of the group 1 received a combination of tetracycline, and streptomycin on alternate days for 2 weeks before mating with a fertile male. An injection of 20µg of gonadotrophin-releasing hormone (Receptal, Intervet) was given to them immediately after mating to induce ovulation. The embryos were collected by the non-surgical method on Day 7 day after ovulation as per the guidelines published by IETS. All embryos were washed at least 3 times in holding medium supplemented with antibiotics and then transferred individually into the left uterine horn of synchronized recipients. All recipients and calves born were tested for brucellosis every 3 weeks. The data were analysed using a two-sample t-test (Minitab statistical software, Minitab Ltd.). Results are shown in Table 1. No difference was observed in the number of embryos collected per flushing and pregnancies established at Day 60. The proportion of pregnancies reaching term from the total number of embryos transferred and from the pregnancies established on Day 60 did not differ from embryos obtained from brucella-infected and control donors. All recipients tested negative for brucellosis during their gestation and until weaning. All calves born were also negative for brucellosis on birth and until weaning and handing over to clients. In conclusion, this is the first study in camels wherein we have demonstrated that invivo-produced embryos from elite dromedary females infected with brucellosis could be collected and transferred to synchronized recipients without transmitting the pathogen to the recipients or offspring. Table 1. Pregnancies established from invivo-produced embryos collected from elite dromedary camels infected with brucellosis Donor group (N) Total number of flushings No. of embryos collected per flushing (n±s.e.m.) Pregnancies on Day 60 (% mean±s.e.m.) Pregnancies reaching term (% mean±s.e.m.) from: Embryos transferred Pregnancies on Day 60 Brucella (10) 30 5.4±0.6 55.1±2.6 44.4±2.3 83.2±3.6 Control (3) 6 5.3±0.6 65.7±6.3 53.3±6.8 80.9±7.7 P-value 0.969 0.173 0.262 0.797

Superovulatory Response and Progesterone Profile in Murrah Buffaloes (Bubalus bubalis) Pretreated with GnRH Agonist

Background: Limited elite buffalo population need to be propagated at faster rate to sustain the dairy industry in India. Faster propagation could be achieved through embryo transfer technology. Low serum progesterone concentrations during the start of superstimulatory treatment is major factor that hampers the success of multiple ovulation embryo transfer (MOET) in buffaloes. This study evaluated the effect of GnRH pretreatment before superovulatory regimen on progesterone profile, superovulation and correlation of progesterone with superstimulatory parameter in Murrah buffaloes. Methods: Buffaloes (n=27) were superstimulated using 600 mg Folltropin and divided into three groups on basis of pretreatment: Group I and group II were pretreated with GnRH @ 10 µg and 06 µg, respectively while no pretreatment was subjected in group III (control). Progesterone hormone concentration, superovulatory response, ovulation rate, embryo recovery were recorded. Result: Our study revealed that superovulatory response was non-significantly different in all three groups and ranged from 6-8 in terms of number of corpus luteum (CL). Average number of recovered embryos in this study was nearly 2.0. A positive correlation between progesterone concentrations at the initiation of FSH treatment with number of CL and embryo recovery was observed. The study indicated that GnRH pretreatment before superstimulation improve the serum progesterone concentration, superovulatory response and embryo recovery in Murrah buffaloes.

Analysis of Genetic Variability in the Argentine Polo Horse With a Panel of Microsatellite Markers

Argentine Polo (AP) is a young horse breed with a fast expansion because of an open registry policy and the application of assisted reproduction technologies. In the last years, AP showed a remarkable progress associated with the use of embryo transfer technology and intensive selection based on sport performance. However, these practices could have affected the genetic variability of the breed. To monitor these potential changes, a parentage panel of 11–15 microsatellites was investigated for changes in allele frequencies, heterozygosity, and exclusion probability over a 16 year period. Frequency of 36 alleles in 13 markers showed significant departures from expected changes because of random genetic drift. Six markers showed both allelic frequency increase and expected heterozygosity (He) reduction, suggesting the influence of selective breeding or hitchhiking effects. The average He decreased significantly although was lower than the observed heterozygosity, indicating a still low inbreeding level. Although the exclusion probability of the panel showed a trend to decrease, it is over 0.9995, the recommended value for equine parentage exclusion.

Blastocyst-Bearing Sows Display a Dominant Anti-Inflammatory Cytokine Profile Compared to Cyclic Sows at Day 6 of the Cycle.

Simple SummaryA proper uterine environment is basic for obtaining optimal embryo transfer outputs in domestic species, including the pig. However, scarce information is available about the uterine immune response of recipient (uninseminated) sows when receiving embryos during embryo transfer. Endometrial cytokine profile is among the main factors regulating uterine receptivity to embryos. In this study, using Luminex MAP® technology, we found important differences in the endometrial production in most of the 16 cytokines analyzed between recipient sows and embryo-bearing (inseminated) sows six days after estrus, with a predominant cytokine anti-inflammatory environment in the embryo-bearing endometria. These observations suggest that insemination components and/or early embryos induce an endometrium immune-tolerant cytokine profile at Day 6 of the cycle. The findings could contribute importantly to design strategies to maximize the reproductive performance of recipients after embryo transfer in swine.In the context of porcine embryo transfer (ET) technology, understanding the tightly regulated local uterine immune environment is crucial to achieve an adequate interaction between the transferred embryos and the receiving endometrium. However, information is limited on the uterine immune status of cyclic-recipient sows when receiving embryos during ET. The present study postulated that the anti- and proinflammatory cytokine profile 6 days after the onset of estrus differs between endometria from uninseminated cyclic sows and blastocyst-bearing sows. On Day 6 of the cycle, endometrial explants were collected from sows inseminated or not inseminated during the postweaning estrus and cultured for 22 h. The culture medium was then analyzed for the contents of a total of 16 cytokines using Luminex MAP® technology. The results showed important differences in the endometrial production of most cytokines between the sow categories, with a predominant anti-inflammatory environment displayed by the blastocyst-bearing endometria. These findings suggest that sperm, seminal plasma (SP) and/or early embryos modify the uterine environment by inducing an immune-tolerant cytokine profile already visible at Day 6. Whether the SP or some of its active components may help to develop strategies to maximize the reproductive performance of recipients after ET needs further investigation.

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation.

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Problematic issues in the effectiveness of embryo transfer technology in cattle

Problematic issues in the effectiveness of embryo transfer technology in cattle

Matrix metalloproteinases improves trophoblast invasion and pregnancy potential in mice

Successful implantation is closely linked to the expression of MMP-2 and MMP-9, which greatly influence the ability of an embryo to degrade the basement membrane of the uterine epithelium, mainly composed of type IV collagen, and invade the uterine stroma. The objective of this study was to determine the effect of MMP-2 and MMP-9 co-transfer with embryos on reproductive performance in mice. Using invasion assay, we tested the effect of MMP-2 and MMP-9 for their ability to support trophoblastic invasion in vitro. We performed co-transfer of MMP-2 and MMP-9 with mouse embryos to 2.5 days post-coitum (dpc) pseudo-pregnant uteri using nonsurgical embryo transfer (NSET) technique and evaluated the pregnancy outcomes. Uterine tissue samples were collected to determine collagen content by Masson’s trichrome staining. Our results showed that in vitro treatment of MMP-2 and MMP-9 significantly promoted both spreading and invasion of mouse trophoblastic cells compared to the non-treated blastocysts. Moreover, embryo transfer results showed that MMP-9 co-transfer enhanced pregnancy outcome inform of live pup rate by degrading the extracellular matrix, collagen, and facilitate embryo implantation. Taken together our findings imply that MMP-9 can regulate trophoblastic cell invasion during preimplantation, which may have important consequences on embryo implantation, and shed the light on new strategies to avoid miscarriage and provides a platform for successful human embryo transfer technologies.

Birth of first foals through embryo transfer after artificial insemination using frozen semen in Japan

Until now, there have been no reports of foals born through embryo transfer after artificial insemination using frozen semen in Japan. The aims of this study were to develop a riding crossbred horse and evaluate the prospects of embryo transfer technology in multiplying horse population. In both donor and recipient mares, luteolysis was induced by the administration of 0.1 mg Cloprostenol to synchronize the onset of estrus, and ovulation was induced by administering 2000 IU human chorionic gonadotropin (hCG) or 0.75 mg Deslorelin. Frozen semen from an Irish Connemara pony stallion was used to breed a Hokkaido native pony mare by deep-horn artificial insemination (dose, 400 × 106 sperm). A non-surgical technique was used to collect embryos from the donor mare at day 7 post-ovulation and transfer them transcervically into the uterus of recipient mares (n = 4) immediately after collection. Weekly blood samples were collected from the recipients throughout pregnancy. A total of four embryos were recovered from seven collection attempts (57% recovery) from a donor mare in a single breeding season. Three of the four transferred embryos maintained successful pregnancy and delivered a healthy live foal (75% birth). A normal progesterone profile was observed throughout gestation in recipient mares. In conclusion, for the first time, to the best of our knowledge, this study describes the birth of foals through non-surgical transcervical embryo transfer in Japan after artificial insemination using frozen semen. We expect that this new crossbreed (Connemara pony × Hokkaido native pony) will be a good riding breed.

Dairy cow reproduction under the influence of heat stress.

Dairy farming is vulnerable to global warming and climate change. Improving and maintaining conception rates (CRs) have a paramount importance for the profitability of any dairy enterprise. There is an antagonistic relationship between fertility and milk yield, and intensive selection for milk yield has severely deteriorated reproductive efficiency. Irrespective of geography and husbandry, modern dairy cows experience heat stress (HS) effects leading to fertility declines, but it worsens in tropical climates. The threshold of HS experience among modern dairy cow has lowered, leading to decreased thermal comfort zone. Studies show that this threshold is lower for fertility than for lactation. HS abatement and robustness response to lactation yield lead to negative energy balance, and cow's reproductive requirements remain unfulfilled. The adverse effects of HS commence from developing oocyte throughout later stages and its fertilization competence; the oestrus cycle and oestrus behaviour; the embryo development and implantation; on uterine environment; and even extend towards foetal calf. Even cows can become acyclic under the influence of HS. These harmful effects of HS arise due to hyperthermia, oxidative stress and physiological modifications in the body of dairy cows. Proper assessment of HS and efficient cooling of dairy animals irrespective of their stage of life at farm is the immediate strategy to reduce fertility declines. Other long- and short-term mitigation strategies to reduce fertility declines during HS include feeding care, reducing disease and mastitis rates, using semen from cooled bulls, timed artificial inseminations (AI), allied hormonal interventions and use of embryo transfer technology. Ultimate long-term solution should be well-planned breeding for fertility improvement and HS tolerance.

Achievements and future perspectives of embryo transfer technology in pigs.

Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non-surgical ET and short- and long-term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20years. This review includes several aspects of non-surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short-term use in pig production.

Determination of the relationship between serum anti-Müllerian hormone level and superovulatory response in Simmental cows.

The most significant focal points of the embryo transfer technology are as follows: the selection of donors, the response of the selected donor to the superovulation protocol and the obtained number of the transferable embryos. For this purpose, it is suggested that donor selection can be done by anti-Müllerian hormone (AMH) levels, and embryo production is evaluated. AMH is secreted by the granulosa cells of primordial, pre-antral and antral follicles below 4mm in the ovary, independent of FSH. Therefore, the aim of this study was to investigate the relationship between serum AMH levels and the number of corpus luteum (CL), total embryos and transferable embryos that were shaped after a uniform superovulation protocol. For this reason, 48 Simmental cows, which were located at General Directory of Agricultural Enterprises (region, province, etc. instead of the general directorate), were used as donors for the embryo transfer. Blood samples were taken at random, regardless of the stage of animal's sexual cycle. AMH levels were measured by enzyme-linked fluorescent assay (ELFA) method of the miniVIDAS® (bioMérieux SA) using AMH Bovine Test Kit. According to the statistical analyses of the obtained data, AMH levels were positively correlated with CL and total embryos (p<.05). No significant correlations between AMH and transferable embryos were approved (p>.05). It was also determined that each 200pg/ml increase in serum AMH level resulted in one increase in CL number. Overall, considering the positive correlation between AMH level and the obtained number of CL and total embryos after a superovulation treatment, it was concluded that measuring blood AMH level prior to any further costly implementation may be an effective method in donor selection.