In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes

To develop a reliable follicle culture system, mouse preantral follicles 150-200 microm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.     

哺乳动物腔前卵泡体外培养研究进展

哺乳动物腔前卵泡的体外培养可充分挖掘优良母畜潜在的遗传和繁殖潜力,为体外胚胎生产提供大量的卵母细胞来源,因而正日益受到人们的重视。本文就哺乳动物腔前卵泡的体外发育中的培养系统、培养方法,培养形式以及影响其体外发育的因素等几个方面进行了阐述。     

家畜卵母细胞冷冻保存的研究进展

本文阐述了卵母细胞冷冻保存对体外受精、转基因动物研究及人类医疗方面的意义,论述了卵母细胞冷冻保存的发展过程、方法与机理及冷冻保护剂种类,通过揭示冷冻、解冻对细胞的作用,进一步探讨了各种冷冻保护剂及冻存方法对保持细胞生命力的功效。在当前卵母细胞冷冻研究中,玻璃化冷冻法发展迅速,并出现超快速玻璃化法。     

家畜胚胎着床的分子调控

胚胎着床的成功进行,需要胚胎和母体之间进行密切的分子对话和精密的信号协调.母体和胚胎来源的类固醇激素、生长因子、细胞因子、整合素及其配体,在家畜的胚胎着床及其母体妊娠识别过程中起着重要的作用.     

Fibrin–alginate hydrogel supports steroidogenesis,in vitro maturation of oocytes and parthenotes production from caprine preantral follicles cultured in group

This study aimed to establish a culture system that improves the in vitro development of caprine preantral follicles. In a first experiment, follicles were encapsulated as a single unit per bead and cultured singly or in groups or with five follicles in the same alginate (ALG) bead for 18 days. In a subsequent experiment, the “five follicles per bead” design was chosen to culture in ALG, fibrin–alginate (FA) or hyaluronate (HA) for 18 days. In a third experiment, we chose the five follicles per bead in FA to culture for 30 days. The culture set‐up of five follicles per ALG bead increased antrum formation and follicle diameter compared to the other culture designs (p < .05). Moreover, under this condition, 44.44% of the oocytes from in vitro cultured preantral follicles reached meiotic resumption. A significant increase of follicle diameter occurred in attachment system and FA (p < .05), but the ALG condition reached the highest among all groups on day 18 (p < .05). Follicles encapsulated in matrix produced more estradiol and progesterone than attachment system (p < .05). The expression of MMP‐9 mRNA was higher in FA than in other groups (p < .05) and similar to antral follicles from in vivo control (p > .05). Only FA group resulted in oocytes matured. After 30 days, oocytes from preantral follicles in vitro grown in FA developed to eight‐cell parthenotes. In conclusion, a culture system using FA supported the development of caprine preantral follicles cultured in group and included in the same bead of hydrogel, improving the oocyte maturation and producing parthenotes.     

Allometric study on the relation between the growth of preantral and antral follicles and that of oocytes in bovine ovaries

We examined the relation between the growth of preantral and antral follicles and that of their oocytes in the ovaries of Holstein cows. We recovered follicles and oocytes (419 pairs) from the ovaries of 61 cows, and examined the relative growth relating the follicle diameter to the oocyte diameter by using six regression models for only healthy oocytes and all the oocytes including degenerated ones with and/or without zona pellucida. The best fitting model was found to be a hyperbolic regression (R(2): 0.999). The differentiated equation for the hyperbolic curve in normal oocytes with zona pellucida and the follicles was found to be y'=41.0/(x+0.253) (2): y and x are diameters of oocytes (microm) and follicles (mm), respectively. When follicles grew more than 4.0 mm in diameter, the growth rate of the oocytes calculated by the differentiation equation was found to be an asymptotic depression around zero. Thus, it is suggested that when the follicles grow more than 4.0 mm in diameter, the oocytes reach full size and cease to grow. Furthermore, it is considered that the equation can be applied to the assessment of normal growth in oocytes and follicles cultured in vitro.     

幼畜与成年畜超排卵发育潜能的研究进展

诱导幼畜超数排卵技术可以充分挖掘和利用优良母畜的早期繁殖潜力,大大加快家畜育种及品种改良进程,同时也将解决种畜胚胎移植商业化所需胚胎来源匮乏及胚胎生产成本昂贵的问题,将大大促进胚胎生产的商业化应用,前景十分广阔。但是,这种幼畜超排的卵,在体外受精后,发育到囊胚期的潜能比成年动物超排的卵发育潜能有所降低,例如,犊牛和成年牛,羔羊和成年羊中就存在此类现象。这种差异可能具有普遍性。本文就幼畜与成年畜超排卵发育潜能的差异做了综述。     

Explanted embryo culture: In vitro and in ovo techniques for domestic fowl

1. Embryos of the domestic fowl (72 h old) have been explanted into shell‐less cultures or ‘surrogate’ eggshells, in order to investigate the possibility of rearing these embryos to hatching.

2. Rocking embryo cultures during the first half of incubation enhanced embryo growth.

3. Embryos explanted into ‘surrogate’ eggshells of either other individuals or other species have been successfully ‘hatched’.

4. A normal chorioallantois is formed in these surrogate eggshells. This enables a functional albumen sac to form and eggshell resorption to be achieved.

5. Embryos grown in ‘surrogate’ eggshells are slightly smaller than controls but otherwise normal.

6. The technique provides opportunities for genetic engineering experiments.     

Isolation of dermatophytes from domestic animals in Norway

The examination of 2066 skin scrapings from domestic animals in the period from July 1981 to June 1984, revealed dermatophytes in 439 samples. Dermatophytes isolated were: M. canis in dog, cat, cattle, horse, swine, goat, rabbit and hamster, M. equinum in dog and horse, M. gypseum in horse, T. equinum in horse and cattle, T. mentagrophytes in dog, cat, cattle, horse, guinea pig and rabbit, T. verrucosum in cattle and E. floccosum in dog.     

Granulosa cells in three-dimensional culture: A follicle-like structure for domestic cat vitrified oocytes

Follicle-like structures are three-dimensional matrices joint with living cells that allow the in vitro development of female gametes in more physiological conditions. They have been shown to be beneficial to fresh oocytes in different species, and in this study, domestic cat (Felis silvestris catus) granulosa cells were used to create a functional follicle-like structure aimed at supporting the in vitro maturation of conspecific vitrified oocytes, key players of fertility preservation programmes that usually struggle to acquire their full developmental competence after warming. Cat granulosa cells were cultured for up to 6 days in three-dimensional barium alginate microcapsules (i.e. follicle-like structures) or in two-dimensional monolayers, and their steroidogenic ability (estradiol and progesterone secretion) was assessed to confirm their functionality. The same systems were used (on day 2 or 6 of granulosa cells culture) for the in vitro maturation (IVM) of Cryotop^® vitrified immature cat oocytes and compared with microdrops of IVM medium without cells (control). Granulosa cells were able to maintain their functionality in vitro in both the conditions, even if with a different extent of hormonal secretion along culture (p = .02). Vitrified oocytes resumed meiosis at higher rates when cultured with 2 days old granulosa cells (p = .03), but full maturation rates slightly raised when granulosa cells were cultured longer, albeit without differences with the control group. This study paved the road for the creation of enriched culture systems in the domestic cat, but innovations are strongly needed for vitrified oocytes that deserve better chances to develop in vitro.     

体外培养牛腔前卵泡卵母细胞微绒毛的超微结构观察

牛腔前卵泡在无血清下体外培养 1 5d。超微结构研究显示 :培养前腔前卵泡卵母细胞微绒毛短小 ,分布均匀 ;培养 6d时 ,微绒毛略变粗长 ,但数量减少 ;培养 1 5d时 ,微绒毛增多 ,变长 ,斜向或垂直插入已经形成的透明带中。微绒毛的存在和变化表明腔前卵泡在该无血清体系中发育正常。     

In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to develop to the blastocyst stage.

Two groups of mouse preantral follicles with diameters of 125-150 and 151-175 microm were cultured individually for 6 days in a medium supplemented with FSH and fetal calf serum to determine their in vitro growth characteristics. Their oocyte capacity for maturation and development to the blastocyst stage following in vitro fertilization was also assessed. Antral formation rate at the end of culture was higher in the follicles of 151-175 microm (89%) than 125-150 microm (76%). The timing of antrum formation was different between the two follicle categories: most 151-175 microm follicles formed antra earlier than 125-150 microm follicles (days 4 and 5 vs. 5 and 6). However, follicle diameters at the time of antrum formation were the same regardless of the initial size and the culture period. Maturation rates of the oocytes derived from both categories of in vitro grown follicles (70 and 62%) were not different from those of oocytes from in vivo grown follicles (74%). The in vitro derived oocytes, however, showed less cleavage (30 and 35%) than the in vivo derived oocytes (89%). Although the oocytes from both follicle categories developed to the morula stage after in vitro fertilization, blastocysts were only obtained from oocytes derived from the 151-175 microm category. These results demonstrate that an individual follicle culture system using a medium with FSH and fetal calf serum supports in vitro growth of mouse preantral follicles with diameters of 151-175 microm to the preovulatory stage, and that their oocytes have the capability to develop to the blastocyst stage.     

Live offspring from cryopreserved embryos following in vitro growth, maturation and fertilization of oocytes derived from preantral follicles in mice

This study was undertaken to examine pre- and postimplantation developmental potency of cryopreserved embryos that had undergone in vitro growth (IVG), maturation (IVM) and fertilization (IVF) of oocytes from the preantral follicle stage. An oocyte culture system for IVG and IVM was used in oocyte-granulosa cell complexes (OGCs) derived from preantral follicles in 12-day-old mice. The rate of oocyte maturation was improved by the addition of gonadotropins (FSH/LH) and cytokines (IGF-I/SCF) to culture medium for IVG. During culture for IVG, estradiol-17β and progesterone concentrations increased progressively to the latter period of culture. This culture system enabled IVG, IVM, IVF and pre- and postimplantation development. From 90 cryopreserved 2-cell stage embryos transferred into recipients after warming, 10 live pups were produced. Cryopreservation of embryos by vitrification at the 2-cell stage showed no harmful effect on development to the blastocyst stage or on the cell numbers of the inner cell mass (ICM) and trophectoderm (TE). This study demonstrated that embryos derived from oocytes grown in vitro have tolerance for vitrification and competence to develop to term after warming. This IVG-IVM-IVF technology combined with embryo cryopreservation might be useful for assisted reproduction in mice.     

水牛腔前卵泡的分布及形态学研究

用常规石蜡切片法研究水牛卵泡在卵巢中的分布规律与结构特征。结果表明各级卵泡的分布具有明显的区域性;原始卵泡位于卵巢皮质的最外层,形成原始卵泡层,初级卵泡位于皮质的中层,次级卵泡位于富含血管的皮质最内层;原始卵泡、初级卵泡和次级卵泡的直径分别为(36.9±7.7)μm、(48.7±8.9)μm和(91.9±27.3)μm,与之对应卵母细胞的直径分别为(19.6±6.0)μm、(27.3±7.1)μm和(49.1±17.4)μm;原始卵泡和初级卵泡的颗粒细胞数为(10.8±2.3)个和(18.1±3.1)个;次级卵泡含有2~6层颗粒细胞,2~4层的颗粒细胞分布不均。     

Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles

This study evaluates the effects of ascorbic acid and its interaction with follicle-stimulating hormone (FSH) on the morphology, activation, and in vitro growth of caprine preantral follicles. Ovarian fragments were cultured for 1, 7, or 14 d in minimum essential medium (MEM) containing ascorbic acid (50 or 100 μg/mL), FSH (50 ng/mL), or both of these substances. Ovarian tissue that was either fresh (control) or cultured for 1, 7, or 14 d was processed for histological and ultrastructural evaluation. The results showed that after 14 d of culture, medium supplemented with 50 μg/mL of ascorbic acid alone or combined with FSH showed higher rates of follicular survival compared with MEM. After 7 d of culture, FSH, ascorbic acid at 50 μg/mL with or without FSH, and ascorbic acid at 100 μg/mL increased the percentage of follicular activation compared to fresh control. In addition, FSH alone significantly increased the percentage of growing follicles after 14 d. The combination of 50 μg/mL of ascorbic acid and FSH promoted a significant increase in oocyte and follicular diameter after 7 d of culture. Ultrastructural and fluorescent analysis confirmed the integrity of follicles cultured with 50 μg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50 μg/mL of ascorbic acid and FSH maintained follicular integrity and promoted follicular activation and growth after long-term in vitro culture of caprine preantral follicles.     

Evaluation of in vitro culture systems for goat preantral follicles using reused ovaries from reproductive biotechniques: An alternative to maximize the potential of reproduction

This study aimed to examine the in vitro culture of secondary preantral follicles, using reused ovaries, to compare both the 2D and 3D methods of in vitro culture of preantral follicles, and the system of medium replacement. Twenty‐five pairs of ovaries from mixed‐breed goats were used for the experiment. Follicular puncture of antral follicles was performed for in vitro production. After this procedure, the secondary preantral follicles were submitted to a microdissection procedure. The isolated preantral follicles were randomly divided into three treatments: (a) Two‐dimensional culture with partial replacement of medium during culture (2D PR), (b) Three‐dimensional culture with addition of medium during culture (3D AD) and (c) Three‐dimensional culture with partial replacement of medium (3D PR). The culture period was 18 days. All treatments at the end of the in vitro culture period (18 days) presented a follicular survival rate which ranged from 59% to 70%, demonstrating that it was possible to perform an experiment with preantral follicles using ovaries that had previously been used in another reproductive biotechnique. The 3D AD treatment showed a survival percentage and follicular diameter higher than the 2D PR treatment, however, it did not differ from the 3D PR treatment. In conclusion, experiments employing the use of preantral follicles can be performed with success after the ovaries have been used for experiments with antral follicles. Moreover, the three‐dimensional system with the addition of medium is recommended for in vitro culture of preantral follicles, since this system is more practical and financially feasible.     

Isolation of obligate anaerobic bacteria from ulcerative keratitis in domestic animals

Objective To determine the frequency of obligate anaerobic bacterial isolation from corneal samples of domestic animals with ulcerative keratitis and to characterize the historical, clinical, cytological, and microbiological features of culture‐positive cases. Animals studied Three hundred and thirty domestic animals with ulcerative keratitis. Procedures Anaerobic bacteriologic culture and Gram stain were performed on corneal samples from consecutive animals examined with suspect septic ulcerative keratitis. Additional corneal diagnostics included: aerobic bacteriologic culture for all species; fungal culture for ungulates; Mycoplasma culture and virus isolation or feline herpesvirus‐1 (FHV‐1) polymerase chain reaction (PCR) for cats. Historical, clinical, and cytological findings were correlated with microbiologic data. Results Anaerobic bacteria were isolated from 13.0% of corneal samples (dogs: 14.0%; horses: 12.9%; cats: 7.9%; alpacas: 18.8%). The most frequent isolates were Clostridium, Peptostreptococcus, Actinomyces, Fusobacterium, and Bacteroides species. The majority of these infections were mixed anaerobic and aerobic bacteria, unless antimicrobial therapy had been administered prior to presentation. The clinical appearance of anaerobic bacterial culture‐positive cases was highly variable. Ocular trauma, pre‐existing corneal disease, previous corneal surgery, and chronic dermatological disease were significantly (P ≤ 0.05) correlated with positive anaerobic cultures in one or more species. Conclusions The results of the present study demonstrate that obligate anaerobic bacteria are present within the intralesional flora of ulcerative keratitis in domestic animals. In most species evaluated, these bacteria were identified infrequently. Anaerobic bacterial infection of the cornea most frequently occurs in association with other ocular pathogens and previous corneal abnormalities.