The duration of follicles’ formation is different among species. The length is ranged 4-6 months from primordial follicle stage to ovulation in sheep and cattle and is shorter in pig
(Campbell et al., 2003; Hunter et al., 2004; van den Hurk and Santos, 2009). Competencies of oocytes are acquired during follicle formation
(Albertini, 2015). At the onset of follicular phase, there is a pool of vesicular follicles from which the ovulatory follicles is continuously selected thereafter. It has been estimated that an early antral follicle in cattle takes about 40 days to develop to the preovulatory follicle. Follicular wave emergence is preceded by an increase of follicle stimulating hormone
(Webb et al., 2003). Granulosa cells around the time of follicular selection acquire LH receptors that are essential for further development (
Baird and Mitchell, 2013). Receptors of LH hormone increase as follicle grows in both granulosa and theca cells. Mature ovulatory follicles are characterized by high expression of aromatase in granulosa cells, high concentration of estradiol in follicular fluid and acquisition of LH receptors on granulosa cells
(Webb et al., 2003). Ovulation occurs in sheep and cattle within 24 hrs. after LH surge. Follicular diameter and FF composition are affected by follicular development and/or level of nutrition (Fig 1) during estrous cycle. This in turn affects oocyte maturation and subsequent embryo development. Diameters of gonadotrophin dependent follicles in some species are illustrated in Table 1.
Follicular fluid composition changes during estrous cycle due to follicular development and/or level of nutrition. High metabolic activity occurs during the preantral and early antral stages of follicular development such as rapid expansion of granulosa cells and increase of oocyte diameter and its protein content. Oocytes are kept in meiotic arrest until LH surge to resume meiosis from dictate germinal vesicle (GV) stage to metaphase II (MII) stage. Similar changes in FF composition are necessary for oocyte growth, maturation and embryo development. Some components, which are known to influence oocyte maturation and/or embryo development are affected by follicular growth and development and/or by level of nutrition, whereas others are still need further investigation (Fig 1). Therefore, the trials to improve culture conditions (
Mohammed 2006;
Mohammed 2008;
Lee et al., 2018) for
in vitro embryo production (IVP) are still required as quality of oocytes (
Mohammed 2017;
Mohammed and Al-Hozab, 2018;
Maylinda et al., 2018; Raj et al., 2018).
Effects of follicular fluid on oocyte maturation in vitro
Duration of oocyte maturation
in vitro from germinal vesicle to metaphase stage lasts in most species 17 to 48 hrs
(Mohammed et al., 2008; Moulavi and Hosseini, 2018). Oocyte maturation
in vitro (IVM) is influenced by the addition of FF to maturation media
(Valckx et al., 2015). The effect is dependent on the size of follicle from which the FF was obtained
(Oberlender et al., 2013), timing of follicular fluid collection either before or after LH surge
(Romero-Arredondo and Seidel, 1994) and percentage of FF added to maturation media
(Mohammed et al., 2005) in different species Table 2.
Inhibitory effect
There are several studies investigated the potential roles of FF on maturation of oocytes and development of the resulting embryos, which collected according to follicle size, or luteinizing hormone (LH) surge. Percentage of FF supplemented to maturation media was ranged from 5-100%. The inhibitory effects of FF on oocyte maturation were described in different species
(Tsafriri et al., 1976) and human as well.
Dostál and Pavlok (1996) found that bovine follicular fluid collected from follicles with diameters of 2.5-5.0 mm showed higher meiosis inhibiting effect on maturation of oocytes
in vitro than that collected from follicles with diameter 5.0-10.0 mm. The effect of supplementing maturation media with 20% bovine FF (bFF) collected from follicles of < 2mm, 3-7 mm, 8-15 mm and >15 mm diameter was investigated
(Wang et al., 1999). Embryo developments were significantly lower in the maturation medium supplemented with bFF from small follicles (< 2mm, 3-7 mm) than that supplemented with bFF from large follicles (> 15 mm). It has been found that FF obtained from follicles smaller than 3 mm had the strongest inhibitory effect on the resumption of meiosis
(Goncalves et al., 2001). bFF collected at 0 or 4 hr following LH surge appeared to possess a meiosis-inhibiting activity that is absent from follicular fluid collected at 8 hrs or more after LH surge (
Romero-Arredondo and Seidel, 1994). Coelho
Cruz et al., (2014) investigated maturation and embryo development upon adding bovine follicular fluid to maturation medium (25, 50, 75, 100%) which collected from follicle with diameter 8-15 mm. They found that high FF concentration (75 - 100% FF) slowed meiotic progression and cortical granule migration. It could be concluded that FF obtained from smaller follicles has inhibitory effect on oocytes’ maturation than that obtained from larger ones.
It has been indicated that the mammals’ oocytes are arrested in the diplotene stage of first meiotic division. Two mechanisms in meiotic arrest were suggested: firstly, the FF was mediating the inhibitory action on meiotic resumption (
Meinecke and Meinecke-Tillmann, 1981) or secondly close contact of the oocyte with follicular cells for an inhibitory signal to be transferred
(Racowsky et al., 1989). Based on these findings, meiosis-inhibiting substance(s) are secreted by granulosa and/or theca cells and require compact cumulus association with oocytes to be transferred and maintain meiotic arrest.
Inhibitors of meiotic resumption in FF were described in several studies. The oocyte maturation inhibitor (OMI) described by
Tsafriri et al., (1982) was protein secreted by granulosa cells and was found to be not species-specific. Porcine oocyte maturation inhibitors is heat stable and its activity was arrested by trypsin
(Tsafriri et al., 1976). These results led the authors to conclude that oocyte maturation inhibitor is probably a peptide.
Dostál and Pavlok (1996) showed that bovine oocyte maturation inhibitors have different properties than that of porcine oocyte maturation inhibitors. Another factor, which isolated from bovine FF (bFF), blocks the progesterone, induced maturation of xenopus oocytes (
Kadam and Koide, 1991). This factor has a molecular weight of 8 kDa. Hypoxanthine, which found in porcine FF (pFF) or bFF, has negative effect on mouse oocyte maturation (
Kadam and Koide, 1990). Hypoxanthine maintains high levels of cyclic adenosine monophosphate (cAMP) within the oocyte
(Downs et al., 1989). Cyclic adenosine monophosphate has been proposed to be the inhibitory signal that maintains bovine oocytes in the germinal vesicle stage of nuclear maturation.
Stimulatory effect
In most species, oocytes resume meiosis after LH surge and ovulate at metaphase II stage. FF has been used as supplement during
in vitro maturation to stimulate oocyte maturation and further embryo development.
Hunter et al., (1973) observed a higher incidence of bovine oocyte maturation in a medium containing follicular fluid.
Chauhan et al., (1997) replaced serum and hormone supplements with FF in the maturation media and they found that nuclear maturation rates of oocytes were similar between groups. In a study of
Algriany et al., (2004), they found that supplementing maturation medium of porcine cumulus-oocyte complexes with FF collected from large follicles enhanced induction of cumulus expansion of COCs, increased nuclear maturation of COCs and their competence to develop to blastocyst stage compared to COCs matured in the presence of FF collected from small follicles.
Oberlender et al., (2013) supplemented maturation medium of porcine oocyte with pFF collected from small (S) and large follicles (L). They found that maturation rate was significantly higher in oocytes matured in a medium supplemented with LpFF than that supplemented with SpFF.
Other studies were carried out using follicular fluid collected during estrous cycle or after LH surge. Bottcher
et al., (1992) found that addition of 20% bFF obtained from estrous cow during maturation increased embryo yield over that found with fetal calf serum (FCS) or bFF from non-estrous cows.
Romero-Arredondo and Seidel (1996) found that bFF collected at 8 hrs. or more after LH surge possess a stimulatory effect on oocyte maturation and embryo development. Pure equine preovulatory follicular fluid collected after gonadotropin-priming was superior in supporting nuclear maturation to standard culture media
(Bøgh
et_al2002).
It is not clear yet to which factor(s) this phenomenon can be attributed.
Kato and Seidel (1998) presented evidence that the composition of bFF changed between 8 and 20 hrs. after the LH surge and it contained a factor(s) by 20 hrs. after LH surge that stimulated the resumption of meiosis.
Collins and Wright (1995) supplemented maturation media with 20% bFF collected from follicles >15 mm which was centrifuged or filtered or heated at 56°C for 30 min. The results of this experiment indicated that factors found in bFF that improve maturation of bovine oocyte might include heat-labile proteins inactivated by heat treatments or factors removed by filtration. It has been suggested that follicular factors derived from granulosa and/or theca cells and accumulated in FF at later stages of follicular development supported developmental competence of oocytes. Indeed, higher nuclear maturation has been obtained of procine COCs matured in medium conditioned with large follicular shells than of COCs matured in medium conditioned with small follicular shells (
Ding and Foxcroft, 1994).
On the other hand, FF collected from small follicles had significantly higher potential of developing bovine
(Choi et al., 1998) and bubaline oocytes
(Nandi et al., 2008) to embryonic stages
in vitro than that from large follicles, and those studies are controversial.
Collection of follicular fluid from live animals or slaughterhouse ovaries
The ability of FF to support cumulus-oocytes complexes during
in vitro maturation appears to be influenced by its origin. Because oocytes resume meiosis after LH surge, it would be logical to consider follicular fluid collected after LH surge favorable for oocyte maturation and embryo development.
Romero-Arredondo and Seidel (1994 and 1996) concluded that bFF from the preovulatory follicle at precise times after LH surge enables more physiological relevant influences than bFF from follicles with unknown history.
Sirard et al., (1995) stated that the factors involved in increased development of oocytes are not necessarily present in all large follicles but seems to be expressed in selected large follicles.
Carolan et al., (1996) observed that the effect of follicular fluid on cytoplasmic maturation of bovine oocytes differs with follicle quality but not size. Our results
(Mohammed et al., 2005) demonstrated that careful collection of pooled bFF from highly vascularized large follicles of slaughterhouse ovaries irrespective of estrous stage resulted in repeatable results in blastocyst rate and quality in different collection batches. In a recent study,
Algriany et al., (2004) investigated nuclear maturation of porcine cumulus oocyte complexes (COCs) cultured in pFF, which collected from three different batches of slaughterhouse ovaries and they did not find any significant differences between batches. Similar result was obtained by
Coelho Cruz et al., (2014) using bovine follicular fluid. Collectively, these studies given experimental background to the hypothesis that follicular fluid collected from large and highly vascularized follicles of slaughterhouse ovaries support developmental competence as FF collected from
in vivo.
As discussed, FF composition changes as follicles develop because of vascularity. It is generally accepted that development of follicle is dependent on angiogenesis
(Koos, 1989). Several studies have been indicated that a dominant follicle has a greater vascular bed and increased of gonadotrophin uptake compared with other antral follicles.
Acosta et al., (2003) demonstrated an increase of both vascular area and blood flow in the pre-ovulatory follicle during spontaneous ovulation.
Van Blerkom (1998) suggested that perifollicular blood flow might be a sign for evaluating quality of oocytes and developmental competence of the resulting embryos. Moreover,
Chui et al., (1997) concluded that follicular vascularity has a possible link with implantation potential.
Kim et al., (2004) suggest that follicular vascularity has a possible link with follicular fluid composition. Therefore, FF could be collected from highly vascularized large follicles of slaughterhouse ovaries. The collected FF can be used during IVM of oocytes in a commercial embryo production program as a cheap material with advantages of blastocyst rate and quality.
Angiogenic factor and follicle development
The principal angiogenic factor that control follicular angiogenesis is likely to be vascular endothelial growth factor (VEGF). FF concentration of vascular endothelial growth factor is higher than in serum
(Artini et al., 1998). Hao et al., (2014) found that vascular endothelial growth factor expression correlated with micro vessel density in the antral follicle of ovine ovary. The exact regulation of vascular endothelial growth factor expression in the follicle is not well known, although
in vivo and
in vitro studies have indicated that gonadotrophins can stimulate granulosa cell production of vascular endothelial growth factor. It has been indicated that administration of vascular endothelial growth factor antagonist prevented the pre-ovulatory follicles development. This inhibition was associated with a decreased thecal layer vascularity, granulosa cell proliferation, antral formation and steroidogenesis
(Zimmermann et al., 2003). Friedman et al., (1998) observed a correlation between the FF concentration of vascular endothelial growth factor and IVF outcome. Van Blerkom
et al., (1997) indicated a potential role of vascular endothelial growth factor in perifollicular angiogenesis and in the regulation of intrafollicular oxygen concentration. The authors suggested that vascular endothelial growth factor could be a sign of healthy follicle but not a clinical prognostic marker for the course of assisted reproduction.
Araújo
et_al(2014) indicated that vascular endothelial growth factor was an effective supplement for culture of bovine secondary follicles
in vitro. Vascular endothelial growth factor has been considered as a stimulator of bovine follicular development
in vitro because it provides support for the transition from the primary to the secondary follicle stage (
Yang and Fortune, 2007).
Percentage of follicular fluid supplementation to maturation medium
There are different concentrations of follicular fluid used during
in vitro maturation in different species ranged from 5 to 100% (Table 2).
Chung and Choi (1974) investigated different proportions of FF during oocyte maturation. The authors suggested that even the presence of a small amount of follicular fluid (13%) in the medium might provide enough factors to initiate meiotic division. A study by
Avery et al., (2003) showed very poor blastocyst rate obtained after
in vitro maturation (IVM) in undiluted bFF although most oocytes were in MII stage meaning that rates of MII stage were not predictive for subsequent fertilization and further development. Addition of 10 to 15% bFF from large and healthy follicles (> 15 mm) to the medium of maturation resulted in higher rates of morula, blastocysts and hatched blastocysts compared with high concentrations of FF or control medium
(Elmileik et al., 1995). Elmileik et al., (1999) examined the effect of adding bFF (follicles > 15 mm) in varying concentrations (15-100%) to the maturation medium and the most favorable outcome (blastocyst rate) was with 50% bFF.
Romero-Arredondo and Seidel (1996) examined 10, 20 or 40% concentrations of bFF and obtained similar results in cleavage and blastocyst rate. FF has been successfully employed on occasions as a protein source at 10-20% level
(Carolan et al., 1996). Both 20 and 40% levels of bubaline FF were observed to be effective for cumulus expansion and nuclear maturation, fertilization and subsequent development of bubaline oocytes to morula and blastocysts stages
(Chauhan et al., 1997). Algriany et al., (2004) supplemented IVM medium with 10% pFF, which was positively stimulated oocyte maturation and blastocyst rate.
Mohammed et al., (2005) added 20% bFF to maturation medium and this concentration support embryo development and quality.
Oberlender et al., (2013) supplemented maturation medium for porcine oocyte with 10% FF collected from small (S) and large follicles (L). They found that maturation rate was significantly high in oocytes matured in medium supplemented with LpFF than that supplemented with SpFF.
It could be concluded that FF supplementation to maturation medium at 10%
(Oberlender et al., 2013); 20%
(Mohammed et al., 2005) and 30-50% (
Coelho Cruz et al., 2014) improved development of oocytes. However, 60% or more of FF has a detrimental effect on embryon development
(Choi et al., 1998; Coelho Cruz et al., 2014). We could conclude also that concentrations of FF collected from large and healthy follicle in diluted level (10-50%) during IVM may favorably influence oocyte maturation and embryo development in bovine, ovine and pig, whereas high concentrations of FF in mares is tolerable during IVM and gave positive results.
Effects of follicular fluid over maturation on developmental competence of embryos
Effects of FF over oocytes’ maturation were followed through further embryonic development. First cleave has been used to predict developmental competence and quality of embryos. Some researchers believe that the first embryonic cleavage has major, long lasting effect on the subsequent developmental of the bovine embryos even to the blastocyst stage and is considered to be a good indicator of those embryos suitable for transfer to recipients
(Lonergan et al., 1999). There are different factors affecting first cleavage of zygote such as paternal genetics
(Ward et al., 2001), sex of zygote and presence of glucose in culture media (Peippo and
Bredbacka et al., 1996). Although several studies investigated the effect of follicular fluid on cleavage rate
(Avery et al., 2003), none of them investigated its effect on first cleavage. In our study
(Mohammed et al., 2005) in vitro maturation media were supplemented with 20% FF and/or 10% FCS to investigate first cleavage at 24, 27, 30, 33, 42 and 48 hour post insemination (hpi). In FCS group, the first cleavage was completed until 33 hpi, whereas in the FF and FF+FCS groups, it continued till 48 hpi. It might be possible that FF rescued the ability of retarded embryos to pursue cleavage. It is not clear to which factors(s) this phenomenon can be attributed. Because FF is transudate of serum not identical in addition to follicular synthesized factors secreted from follicular cells, we suggest that first cleavage due to follicular synthesized factors. Further studies are required to investigate effect of FF on first cleavage and embryo development.
Nutrition and follicle development
Follicles’ diameters change during estrous cycle (
Evans et al., 2000) and/or level of nutrition
(Mackey et al., 1999). Several studies indicated that level of nutrition change follicular diameter. Nutritional restriction (0.4 times maintenance) for about 12 days decreases diameter of the dominant follicle
(Mackey et al., 1999). Cattle fed with low energy intake have smaller dominant follicles and more three-wave cycles per estrous compared with animals fed high feed intakes.
Murphy et al., (1991) reported that heifers offered a low dietary intake had reduced size and persistence of the dominant follicle compared with animals offered high-energy intakes. Fat supplementation to cattle increased the number and size of follicles
(Lucy et al., 1990; Beam and Butler 1997).
Cosgrove et al., (1992) found that re-alimentation of feed restricted gilts or extra feed (
Cox, 1997) increased follicular growth and diameter. Energy status is generally considered a major nutritional factor that influences folliculogenesis (
Moonmaneea and Yammuen-art, 2015).
Moreover, feed additives to animal rations as urea and others effect on follicle development, oocyte quality and FF composition
(Mohammed et al., 2011, Mohammed et al., 2012; Mohammed and Al-Suwaiegh 2016;
Mohammed 2017;
Mohammed and Al-Hozab 2020). As discussed, level of nutrition and feed additive were influenced development of follicles and their follicular fluid’ composition. Consequently, further studies are still required to investigate the changes of follicular fluid and their effects on oocyte maturation
in vitro and further embryo development upon levels of nutrition or feed additives.
Concluding thoughts and perspective remarks
Follicle size and level of nutrition are known to affect FF compositions. Studies used FF during
in vitro maturation were carried out irrespective of level of nutrition. Studies are still required to investigate the effects of FF collected under different level of nutrition on
in vitro oocytes’ maturation and their developmental competence to embryos. We could expect from the aforementioned discussion that using diluted concentration of FF (10-50%) during oocytes’ maturation improves their maturation and development of embryos.
