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In this study, we systematically compared the morphological, functional and molecular characteristics of granulosa cells and oocytes obtained by a three-dimensionalin vitromodel of ovine ovarian follicular growth with those of follicles recoveredin vivo. Preantral follicles of 200 µm diameter were recovered and cultured up to 950 µm over a 20-day period. Compared within vivofollicles, thein vitroculture conditions maintained follicle survival, with no difference in the rate of atresia. However, thein vitroconditions induced a slight decrease in oocyte growth rate, delayed antrum formation and increased granulosa cell proliferation rate, accompanied by an increase and decrease inCCND2andCDKN1AmRNA expression respectively. These changes were associated with advanced granulosa cell differentiation in early antral follicles larger than 400 µm diameter, regardless of the presence or absence of FSH, as indicated by an increase in estradiol secretion, together with decreased AMH secretion and expression, as well as increased expression ofGJA1,CYP19A1,ESR1,ESR2,FSHR,INHA,INHBA,INHBBandFST. There was a decrease in the expression of oocyte-specific molecular markersGJA4,KIT,ZP3,WEE2andBMP15 in vitrocompared to thatin vivo. Moreover, a higher percentage of the oocytes recovered from cultured follicles 550 to 950 µm in diameter was able to reach the metaphase II meiosis stage. Overall, thisin vitromodel of ovarian follicle development is characterized by accelerated follicular maturation, associated with improved developmental competence of the oocyte, compared to follicles recoveredin vivo.

Reproductive efficiency is not optimal in high-producing dairy cows. Although many aspects of ovarian follicular growth in cows are similar to those observed in heifers, there are numerous specific differences in follicular development that may be linked with changes in reproductive physiology in high-producing lactating dairy cows. These include: 1) reduced circulating estradiol (E2) concentrations near estrus, 2) ovulation of follicles that are larger than the optimal size, 3) increased double ovulation and twinning, and 4) increased incidence of anovulation with a distinctive pattern of follicle growth in anovular dairy cows. The first three changes become more dramatic as milk production increases, although anovulation has not generally been associated with level of milk production. To overcome reproductive inefficiencies in dairy cows, reproductive management programs have been developed to synchronize ovulation and enable the use of timed AI in lactating dairy cows. Effective regulation of the CL, follicles, and hormonal environment during each part of the protocol is critical for optimizing these programs. This review discusses the distinct aspects of follicular development in lactating dairy cows and the methodologies that have been utilized in the past two decades in order to manage the dominant follicle during synchronization of ovulation and timed AI programs.

Bu çalışma, Eylül 2010 tarihleri arasında Anchorage[Alaska]’da düzenlenen 8. International Symposium on Reproduction in Domestic Ruminants’da bildiri olarak sunulmuştur. Reduced reproductive efficiency has been reported in high-producing dairy cows. Sources of reproductive inefficiency include decreased expression of estrus, increased diameter of the ovulatory follicle and reduced fertility when cows are inseminated after estrus, increased incidence of double ovulation and twinning, and increased pregnancy loss. To overcome some of these inefficiencies, reproductive management programs have been developed that synchronize ovulation and enable effective timed artificial insemination (AI) of lactating dairy cows. Effective regulation of the corpus luteum (CL), follicles, and hormonal environment are critical for optimizing these programs. Recent programs, such as the 5-day CIDR program, Double-Ovsynch, G-6-G, and estradiol benzoate-CIDR programs were designed to more effectively control one or more physiological events. These events include synchronization of a new follicular wave at the beginning of the program, optimization of the circulating progesterone (P4) concentrations and duration of follicular dominance, optimized reductions in P4 and increases in circulating estradiol (E2) concentrations during the preovulatory period, and tightly synchronized ovulation of a follicle of optimal size and fertility for implementation of timed AI. The success of these programs has been remarkable, although there is substantial variability in effectiveness due to environmental, management, nutritional, genetic, and disease factors as well as potential variability in some aspects of reproductive physiology among commercial dairy farms. Future programs will optimize the reproductive physiology while simplifying the protocol implementation and also match specific reproductive management protocols to specific farms and even specific cows (for example primiparous vs. multiparous).

International audience; In vitro maturation (IVM) of bitch oocytes is, to date, a very inefficient process, with common metaphase rates approximately 0-20% and mean degeneration rates approximately 20-30%. In other mammals, meiotic resumption is controlled in the cumulus-oocyte complex by the disappearance of the coupling between granulosa cells and the oocyte. The first aim of this study was to evaluate the influence on meiotic resumption of a mechanical denudation of the oocytes before maturation. The nuclear stage was determined by DNA staining with ethidium-homodimer-2 under confocal microscopy. Denuded (n = 318) and control (n = 378; no mechanical denudation) oocytes had similar degeneration rates (respectively 32.1 vs 28.6%). However, meiosis resumption rates were significantly higher for denuded oocytes (DO; 26.9 vs 17.8%). Secondly, we aimed to evaluate oocytes experiencing spontaneous denudation during the 72 h IVM period. Denuded oocytes, having lost cumulus cells on at least 75% of their perimeter (n = 440), were compared with surrounded oocytes (SO), with 100% of their perimeter surrounded by granulosa cells (n = 860). As above, the nuclear stage was determined by confocal microscopy, but cytoplasmic maturation was also evaluated through transmission electron microscopy. Degeneration rates but also meiosis resumption and metaphase rates were significantly higher for denuded than for SO (9.6 vs 2.8% for metaphase rate). Nevertheless, ultrastructurally, metaphase DO have scarcer organelles unevenly distributed, with smooth endoplasmic reticulum concentrated in aggregates in the cortical zone. Denudation, whether mechanical or spontaneous, is thus an inefficient mean to obtain metaphase II oocytes suitable for in vitro fertilization.