Heterogenous and xenogenous fertilization of in vivo matured equine oocytes

Forty-five in vivo matured equine oocytes were recovered from 63 follicular aspiration attempts (71.4%). HCG did not improve recovery rate (65% — 24/37 for treated vs 81% — 21/26 for nontreated mares). Fifteen oocytes were transferred into the oviduct of inseminated recipient mares (heterogenous fertilization) and 15 oocytes plus equine spermatozoa were transferred into rabbit oviducts (xenogenous fertilization). Ten oocytes (3 fertilized) were recovered from recipient mare oviducts following removal and flushing two days after transfer. Eight oocytes (nonfertilized) were recovered from rabbit oviducts. Oviductal transfer into separate recipient mares of three embryos produced from heterogenous fertilization resulted in two pregnancies. One mare produced a normal live foal and the other mare aborted at 20 days of gestation. Results from these studies suggest that: 1) a reliable method for collection of in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from valuable infertile mares that fail to get pregnant or produce embryos by standard methods.     

Reproductive Parameters of Mangalarga Marchador Mares in a Commercial Embryo Transfer Programme

The objective of this study is to evaluate the reproductive efficiency in donors and recipient Mangalarga Marchador mares in commercial programmes of embryo transfer (ET) and the effects of some reproductive characteristics and ET methodology on conception rates in the recipient mares. A total of 1140 flushing procedures were performed and 830 embryos (72.8%) were recovered. There were no differences between the rates of embryonic recovery in the different breeding seasons (p > 0.05) and 92.8% of the recovered embryos were 8–9 days old. There was no difference in the embryonic recovery regarding the collection order from the first to the ninth embryo collection along the breeding season, as well as among mares inseminated during the foal heat or subsequent cycles (p > 0.05). Pregnancy rates observed in the total period of all reproductive seasons at 15, 30, 45 and 60 days of pregnancy were 73.4, 69.9, 66.7 and 64.5%, respectively. Differences in pregnancy rate and early embryonic loss rates were not observed between embryos transferred immediately after collection (66.8% and 13.5%) and embryos transported at room temperature for periods of <1 h (62.9% and 14.4%; p > 0.05). Pregnancy rates were higher when the interval between ovulations of donor and recipient mares remained between ?3 and ?2 days (p < 0.05), and the lowest rates were observed for intervals of ?6 days (p < 0.05) with intermediary values for intervals of ?1, 0 and +1 (p > 0.05). Embryonic loss rates, however, did not differ between intervals of ovulation’s synchronism between donor and recipient mares (p > 0.05). This flexibilization in the ovulatory synchronism between donor and recipient mares optimizes the use of recipient mares, thus reducing costs and facilitating management of horse breeding farms.     

Lysophosphatidic Acid Synthesis and its Receptors’ Expression in the Bovine Oviduct During the Oestrous Cycle

Lysophosphatidic acid (LPA) is a naturally occurring simple phospholipid which in the bovine reproductive system can be produced in the endometrium, corpus luteum, ovarian follicle and embryo. In this study, we examined the possibility that LPA receptors are expressed, and LPA synthesized, in the bovine oviduct. We found that the concentration of LPA was highest in infundibulum in the follicular phase of the oestrous cycle and was relatively high during the early‐luteal phase in all examined parts of the oviduct. We also documented that LPA synthesis engages both available pathways for LPA production. The autotaxin (ATX) protein expression was significantly higher in the infundibulum compared to the isthmus during the follicular phase of the oestrous cycle. During the early‐luteal phase of the oestrous cycle, ATX and phospholipase A2 (PLA2) protein expression was highest in ampulla, although the expression of LPARs was not as dynamic as LPA concentration in the oviduct tissue, and we presume that in the bovine oviduct, the most abundantly expressed receptor is LPAR2. In conclusion, our results indicate that the bovine oviduct is a site of LPA synthesis and a target for LPA action in the bovine reproductive tract. We documented that LPAR2 is the most abundantly expressed in the bovine oviduct. We hypothesize that in the bovine oviduct, LPA may be involved in the transport of gametes, fertilization and cellular signalling between the oviduct and cumulus–oocyte complex.     

表皮生长因子在雌性哺乳动物生殖过程中的作用

表皮生长因子 (EGF)是表皮生长因子家族成员之一 ,在哺乳动物生殖过程中具有重要的调节作用。在哺乳动物卵巢内有 EGF的表达并调节卵巢的功能活动 ;EGF可促进哺乳动物卵泡发育 ,加速卵母细胞的成熟 ;EGF可促进卵母细胞受精和受精卵发育的能力 ,促进胚泡腔的形成 ;EGF在许多哺乳动物的子宫内均有表达 ,并参与胚胎着床过程的调节。 EGF可能以自分泌或旁分泌的形式参与卵泡发育、卵母细胞成熟、子宫内膜增殖、早期胚胎发育及胚胎着床等过程的调节。文章对 EGF在雌性哺乳动物卵泡发育、卵母细胞成熟、早期胚胎发育、囊胚腔扩展、胚胎着床等过程中的作用进行了概述     

Manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle

Over the last several decades, a number of therapies have been developed that manipulate ovarian follicle growth to improve oocyte quality and conception rates in cattle. Various strategies have been proposed to improve the responses to reproductive biotechnologies following timed artificial insemination (TAI), superovulation (SOV) or ovum pickup (OPU) programmes. During TAI protocols, final follicular growth and size of the ovulatory follicle are key factors that may significantly influence oocyte quality, ovulation, the uterine environment and consequently pregnancy outcomes. Progesterone concentrations during SOV protocols influence follicular growth, oocyte quality and embryo quality; therefore, several adjustments to SOV protocols have been proposed depending on the animal category and breed. In addition, the success of in vitro embryo production is directly related to the number and quality of cumulus oocyte complexes harvested by OPU. Control of follicle development has a significant impact on the OPU outcome. This article discusses a number of key points related to the manipulation of ovarian follicular growth to maximize oocyte quality and improve conception rates following TAI and embryo transfer of in vivo- and in vitro-derived embryos in cattle.     

Assessing the fertility of two mares cloned from the same founder animal

Two cloned mares, produced from the same sample of skin fibroblasts, were bred during four breeding seasons from their second year of age, as embryo donors, in exactly the same conditions, using the same stallions for both cloned mares. The aim of this study was to test the embryo donor potential of cloned mares and to compare the results obtained from two cloned mares of the same mare with other embryo donor mares (n = 31–39 per breeding season) at the same stud. For both cloned mares, 19 embryos were recovered by 43 collection attempts (44%) (7/22 for one; 12/21 for the other), 16 (84%) pregnancies (5/7 for one, 11/12 for the other) were obtained at day 14 post-ovulation (D₁₄), and 12 (3/7 for one; 9/12 for the other) foals were born. One cloned mare was a less efficient donor mare than the other (p < .05), In control donor mares, 623 embryo collections were performed, with a recovery rate (80%—496/623) significantly higher than for cloned mares. The recovery rate in the subpopulation of 2–5-year-old control donor mares (same age of cloned mares) (89%—127/143) and The recovery rate in the subpopulation of 12 control mares bred with the seven same stallions as clones (55%—17/31), were both higher than for cloned mare (p < .05). The success rate of transfer was not different between embryos produced by cloned mares (84%—16/19) and those produced by control donor mares (79%—392/496). However, the foaling rate per embryo collection was significantly lower for cloned mares (28%—12/43) than for control donor mares (52% - 325/623) (p < .05).     

Influence of Cysteamine on In Vitro Maturation, In Vitro and In Vivo Fertilization of Equine Oocytes

The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, IVF or in vivo embryonic development under our conditions. Ovum pick up yields (52%) and maturation rates (control group: 47% and cysteamine group: 55%) were similar to those previously reported. From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and five in the cysteamine group (9%). Those two percentages were not statistically different (p > 0.05). No effect of IVF technique was seen on the success rate (6%) in each group.     

Embryo transfers from mares in athletic competition

The objective of this project was to produce multiple pregnant recipient mares during one year from each of four mares which were in athletic training and competition. Twenty-six embryos were recovered from the four mares in 38 collection attempts. Each embryo was surgically transferred to one of 20 recipient mares. Nine of 10 recipient mares which were transferred to on one occasion were detected pregnant at the one week post transfer pregnancy examination and six of eight mares which were transferred to on two occasions were detected pregnant at one of the one-week post transfer pregnancy examinations. Three of the 15 pregnant recipient mares lost their pregnancies by day 150. The 12 resulting pregnant recipient mares from the four competing donor mares demonstrated that donor mares can remain in athletic competition and at the same time provide viable embryos for embryo transfer.     

Cumulus–Oocyte Communications in the Horse: Role of the Breeding Season and of the Maturation Medium

Horse is a seasonal breeder and information on oocyte quality outside the breeding season is very limited. Ovaries obtained at the slaughterhouse are a convenient but often limited source of oocytes in this species. As the low quantity of ovaries leads to an intensive use of all available material, it would be useful to know whether ovaries collected during the non‐breeding season are suitable for in vitro maturation (IVM). In an attempt to characterize the effect of season on oocyte quality, we investigated the permeability of the gap junctions (GJ) present between cumulus cells and oocytes because of their important role in oocyte growth and maturation. We also compared the effect of supplementing the maturation medium with bovine serum albumin (BSA) or oestrus mare serum (EMS). A total of 645 oocytes isolated from 158 and 154 ovaries collected during the breeding and the non‐breeding season, respectively, were used in this study. Oocytes were matured for 30 h in TCM 199 supplemented either with 10% EMS or with 4 mg/ml BSA. The presence of permeable GJs between cumulus cells and oocytes was investigated with the injection of a 3% solution of the fluorescent dye Lucifer yellow into the ooplasm. No differences in efficiency of oocyte retrieval or oocyte meiotic competence were detected between oocytes collected during the breeding and non‐breeding season. The vast majority (90%) of the oocytes collected during the breeding season had fully functional communications with their surrounding cumulus cells but such communications were completely interrupted in 55.3% of the oocytes collected during the non‐breeding season. During the non‐breeding season, the proportion of oocytes whose communications with cumulus cells were classified as closed or intermediate at the end of maturation was lower in the group matured with BSA than with EMS (71.4 vs 97.7, p < 0.05). The same trend, although not statistically significant, was observed during the breeding season also. The presence of BSA caused an incomplete cumulus expansion during both seasons. Our data indicate that oocytes collected during the non‐breeding season do not show any meiotic deficiency but lack active communication with the surrounding cumulus cells at the time of their isolation from the ovary. No data are available at present for determining the consequences on the developmental competence even if data from other species suggest that this is likely.     

Differences in development of bovine oocytes recovered by aspiration or by mincing.

This experiment was performed to clarify relationships between conditions of bovine ovaries and developmental capacity of the follicular oocytes recovered from them and to compare two methods of oocyte collection, aspiration and mincing. Follicular oocytes with surrounding intact, unexpanded cumulus recovered by follicular aspiration or by mincing of tissue from 24 pairs of ovaries were matured and fertilized in vitro. The number of follicular oocytes recovered from pairs of ovaries averaged 32.1 +/- 3.2, but the number recovered varied greatly among the 24 pairs of ovaries (range, 7 to 71). The overall rate of development to the blastocyst stage was 18% (137/771), and the average number of blastocysts produced from a pair of ovaries was 5.7 +/- 1.1 (range, 0 to 17). No relationships were found between the presence of corpora lutea or large follicles and the proportion of oocytes capable of reaching the blastocyst stage in vitro. However, a positive correlation was observed between the number of oocytes obtained from each pair of ovaries and subsequent in vitro development; the correlation was especially high for oocytes obtained by aspiration. These data suggest that the developmental capacity of bovine follicular oocytes after in vitro maturation and fertilization is correlated to the number of antral follicles aspirated from the pair of ovaries.     

影响牦牛卵母细胞体外成熟的因素分析

牦牛是青藏高原地区的重要畜种,为牧民提供生产和生活资料.但是牦牛的繁殖力较低,通常为2年1胎或3年2胎,选育改良进展缓慢.卵母细胞体外成熟培养作为体外受精和胚胎发育必不可少的步骤,已被应用于牦牛科学研究和繁殖生产上.由于牦牛卵母细胞体外成熟能力远远低于体内,因此完善体外培养条件,提高牦牛卵母细胞体外培养的成熟率,对牦牛...     

Somatic Cell Nuclear Transfer in Horses

The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo -matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.     

Female traits, ovary and follicle characteristics, and the conditional probability of normal oocyte development after superovulation of beef cows.

The proportion of transferable beef embryos obtained after superovulation, follicle aspiration, and in vitro maturation and fertilization has been small. To seek possible explanations, cows on different planes of nutrition were treated with exogenous gonadotropin and oocytes were isolated from their ovaries. The record for each oocyte included characteristics of the follicle, ovary, and cow from which it was obtained and the response to in vitro maturation, fertilization, and development. The sample was used to obtain estimates of the relationships among the variables. The logistic function with the probability of normal development as the dependent variable was the basic equation of the statistical model. When an explanatory variable was itself a result of the biological system, an equation explaining variation therein was added to the model. Had equations representing endogenous regressors not been added to the model a simple, single equation would have represented oocyte development response; given an oocyte at aspiration only one variable, cumulus quantity, was found to condition the probability of normal development directly. However, the complete model included four additional equations: 1) the probability that an oocyte was recovered at aspiration was conditional on the plane of nutritional treatment and progesterone concentration in follicular fluid; 2) cumulus quantity was conditional on the presence on a corpus luteum, follicle size, and progesterone concentration; 3) progesterone concentration was dependent on plane of nutrition; and 4) corpus luteum was conditional on plane of nutrition. The estimated model provided some insight into the complexity of oocyte development response and the role nutrition may play.     

卵丘细胞对卵母细胞IVM及分子机制的研究进展

卵母细胞的成熟是当前胚胎工程及相关动物生物技术的基础及重要环节,对体外受精、转基因、胚胎克隆以及相关生物技术的发展具有重要的意义,本文主要将卵母细胞体外成熟的分子机制、卵丘细胞对卵母细胞成熟影响因素、目前取得成果以及存在的问题等方面进行归纳总结,为进一步的研究提供理论依据。     

卵母细胞去核及卵丘细胞移核方法对猪体细胞核移植的影响

以猪体外成熟卵母细胞周围分离得到的卵丘细胞作为猪体细胞核移植的供核细胞,研究了卵母细胞不同去核方法(盲吸法、活性荧光染色去核法、盲吸法和活性荧光染色法联合去核法)的去核率差异,并比较了卵丘细胞透明带下注核和胞质内注核两种重建胚构建方法的胚胎裂解率、4 8细胞发育率和桑椹胚率的差异。结果表明:(1)以荧光染料染色法的去核率(87.18%)最高,与其他试验组相比差异显著(P<0.05);盲吸法去核所用时间最少(1.2 min/个),与其他试验组相比差异显著(P<0.05)。(2)透明带下注核比卵母细胞质内直接注核的卵裂率(40.11%/29.75%)和4 8细胞率(11.86%/6.81%)高,差异显著(P<0.05);桑椹胚率(1.75%/1.79%)无明显差异(P>0.05)。     

Ovarian function and morphology in the mare after multiple follicular punctures

REASONS FOR PERFORMING STUDY: In the mare, ultrasound-guided transvaginal oocyte recovery and transfer might offer a way to circumvent the demanding procedures of in vitro embryo production. Before clinical application, the possible consequences for subsequent fertility have to be considered. OBJECTIVES: To examine ovarian function and morphology in mares after repeated follicular punctures. METHODS: A total of 14-26 follicular puncture sessions were conducted on each of 4 Norwegian pony mares over a period of 8 years. The ovaries of these mares were recovered by bilateral ovariectomy or at post mortem and subjected to macroscopic inspection and histology. For comparison, ovaries were collected from 7 nonaspirated control mares and processed for histology. RESULTS: In all experimental mares, ovarian function, defined as the ability regularly to ovulate preovulatory follicles and develop corpora lutea, remained normal during their last breeding season. Gross examination and histology showed that normal follicular and corpus luteum development was accompanied by the formation of condensed reparative fibrosis and normal local haemosiderosis of the ovarian stroma in all experimental mares. In one mare, an ovary contained several foci of chronic apostematous oophoritis, while a cystic structure lined with a single layer of epithelial-like cells and surrounded by a cartilaginous capsule was present in the other ovary. CONCLUSIONS AND POTENTIAL RELEVANCE: Repeated follicular aspirations do not hamper future folliculogenesis, ovulation and corpus luteum formation. However, ovarian puncture induces reparative fibrosis in the ovarian stroma and involves a risk of inducing abscess formation within the ovarian tissue which may impair fertility.     

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography–high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.     

The Development and Application of the Modern Reproductive Technologies to Horse Breeding

Although the horse was probably the first animal to experience and benefit from artificial insemination, it trailed the field somewhat with regard to the application of embryo transfer and other oocyte and embryo-related modern breeding technologies. But with a late run it is now back in mid-field and gaining fast on the other large domestic species in the application of the many technological advances of the past 20 years to sound breeding practice. Improvements in extenders and cryoprotectants have resulted in a veritable upsurge in the transport and insemination of cooled and frozen stallion semen, and parallel improvements in ovulation induction and synchrony, exogenous gonadotrophic stimulation of multiple fertile ovulations and simplified, more efficient methods for non-surgical transfer of embryos to recipient mares, coupled with relaxation of breed society registration restrictions, have together contributed to a similar upsurge in the application of embryo transfer to all breeds and athletic types of horses worldwide, with the continuing and notable exception of the Thoroughbred. Although conventional in vitro fertilization remains something of an unjumped fence in equids, other modern breeding technologies like hysteroscopic low-dose insemination, fluorescence-activated sex sorting of stallion spermatozoa, between-species embryo transfer, embryo freezing and bisection, transvaginal ultrasound-guided oocyte collection, intracytoplasmic sperm injection for fertilization (ICSI), gamete intrafallopian transfer (GIFT) and now nuclear transfer (cloning), have all been applied to equids with encouraging success. Cloning, especially, holds enormous promise for the Sporthorse industry to re-create champion geldings in stallion form for breeding purposes.     

牛卵母细胞体外成熟和体外受精技术的研究进展

论述牛卵母细胞体外成熟和体外受精的最新研究进展，包括卵丘卵母细胞复合体、卵母细胞的体内、体外成熟，以及体内、体外的异常成熟和卵母细胞的体外成熟方法，无蛋白质、无血清系统的限定性培养液的研究进展，卵母细胞体外成熟状态与标志的某些理论上的突破；体外受精中精子供体的选择、精子活力和正常形态的选择、冷冻解冻精液的体外获能、精子的体外受精力，以及体外的异常受精；还论述了牛卵母细胞体外成熟和体外受精技术在家畜育种和胚胎克隆等方面的应用前景