Physiological characteristics and effects on fertility of the first follicular wave dominant follicle in cattle.

The first follicular wave emerges soon after ovulation, and its dominant follicle (DF) develops during the first 8-10 days of the estrous cycle in cattle. And, the first-wave DF is a non-ovulatory follicle, because it develops during the first half of the estrous cycle simultaneously with the corpus luteum (CL), which produces and secretes progesterone. Regarding the characteristics of development and the mechanisms of deviation in the DF during the follicular wave, the first-wave DF has been well studied. However, the characteristics of the first-wave DF, such as growth, blood flow in the follicular wall, concentration of sex steroid hormones in the peripheral blood and follicular fluid, amounts of mRNA in granulosa cells, as well as the characteristics of the CL formed after the first-wave DF and the influence of the first-wave DF on fertility (conception rate), have not been well studied. Additionally, the first-wave DF synthesizes and secretes 17β-estradiol (E2), and plasma E2 concentration increases during the early stage of the estrous cycle. Consequently, there is a possibility that the first-wave DF might affect the fertility in cattle. In this review, to provide the new perspective on reproductive physiology in cattle, characteristics of the first-wave DF were examined in detail and its characteristics were compared with that of the second-wave DF. In addition, the locational effects of the first-wave DF and CL on conception rate are discussed.

Introduction

The reproductive performance of lactating dairy cows has been decreasing worldwide over the past few decades (conception rate: 55% vs. 45%, 1950s vs. 1998; calving interval: 13.5 months vs. 14.8 months, 1970 vs. 1999; services per conception rate: 1.62 vs. 2.91, 1972 vs. 1996) [1]. The decline in fertility has been attributed to various factors, such as lower estrous detection, housing systems, herd size, failure of nutritional management, and metabolic and diseased states [2, 3]. To improve the insemination rate, estrous synchronization programs have been used in reproductive management in the dairy and beef cattle industry [4,5,6]. However, to achieve a higher conception rate, we need a much deeper understanding of the characteristics of ovarian physiology and mechanisms of maternal recognition in high-producing dairy cows.

In cattle, the synchronous development of a group of 8–41 small follicles are observed in 2 to 3 times during an estrous cycle, and these groups developing follicles development are called as a follicular wave [7,8,9,10]. Further, the largest follicle that continues to grow during the follicular wave is defined as the dominant follicle (DF), whereas the other subordinate follicles go into atresia [11, 12]. The first follicular wave emerges soon after ovulation (day of ovulation = 0 day) and the first-wave DF grow 8–11 days of the estrous cycle; however, the first-wave DF becomes a non-ovulatory follicle because it develops during the first half of the estrous cycle simultaneously with the corpus luteum (CL) [9] (Fig. 1). In estrous cycles with a two-wave follicular wave pattern, the second wave emerges on 9–11 days of the estrous cycle, and the second-wave DF becomes an ovulatory follicle [13]. On the other hand, with a three-wave follicular wave pattern, the second-wave emerges on 8–9 days of the estrous cycle, and the third-wave emerges 15–16 days of the estrous cycle, and the third-wave DF becomes an ovulatory follicle [13]. Although the timing of luteolysis (two-wave vs. three-wave: 16 days vs. 19 days) and duration of the estrous cycle (two-wave vs. three-wave: 19–20 days vs. 22–23 days) differ between the two-wave and three-wave patterns [13], the first follicular wave emerges soon after estrus and it becomes a non-ovulatory follicle in both of the two-wave and three-wave patterns.

Fig. 1.

Schematic figure of dynamics of dominant follicle (DF) and corpus luteum (CL) during estrous cycle (two-waves of follicular wave pattern).

Regarding the characteristics of DF development and the mechanisms of selection of DF during the follicular wave, the first-wave DF has been well studied [14,15,16,17]. However, the characteristics of the first-wave DF and the influence of the first-wave DF on fertility (conception rate) have not been thoroughly investigated.

In previous studies, the concentration of 17β-estradiol (E2) in follicular fluid and the production of androstenedione (A4) and progesterone (P4) by cultured theca cells were greater in the first-wave DF than in the second-wave DF [18]. In addition, blood flow (BF) in the follicular wall of the preovulatory follicle is greater during the first wave than during the second wave [19]. Therefore, it is hypothesized that the characteristics of the first-wave DF differ in comparison to those of the second-wave DF.

The effects of the first-wave DF on conception rate have not been well investigated. However, the first-wave DF synthesizes E2 [14], and the plasma E2 concentration increases during the early stage (4–5 days) of the estrous cycle [20]. Consequently, there is a possibility that the first-wave DF might affect fertility in cattle.

In this review, we will discuss 1) the characteristics of the first-wave DF, such as growth, blood flow in the follicular wall, amounts of sex steroid hormones and mRNA in the peripheral blood and follicular fluids, and formation of the CL, and 2) the influence of the first-wave DF on fertility, in particular, the relationship between the location of the first-wave DF and CL in the ovary and the effects on conception rate.

Follicle Development, Blood Flow in the Follicular Wall and Plasma E2 Concentration in the First-wave DF

To determine the characteristics of first-wave DF development, the diameter, blood flow in the follicular wall and plasma E2 concentration ware compared with those of the second-wave DF using hormonal treatment (prostaglandin F2α [PGF2α] and gonadotropin-releasing hormone [GnRH]) to induce estrus and ovulation in non-lactating Holstein cows [21].

In this experiment, spontaneous ovulation day (emergence of the first-wave) was defined as Day 0 in the first-wave group. On the other hand, to mimic second-wave emergence, spontaneous ovulation was defined as Day −7, and GnRH was administrated on Day −2 to induce ovulation of the first-wave DF, and we confirmed ovulation on Day 0 (emergence of the second-wave). Then, we administrated PGF2α on Day 6 and GnRH on Day 8 to induce estrus and ovulation in the first- and second-wave DF. Therefore, a similar time axis of the duration of follicular growth and timing of maturation existed between the first- and second-wave of follicular development and the maturation and changes occurring before ovulation were compared between the first- and second-wave DF.

The diameter of the first-wave DF was larger on Day 6 than on Day 3; however, the diameter of the second-wave DF was not significantly different between Days 3 to 6. After PGF2α treatment, the diameter of the first-wave DF increased between Days 6 and 8, but no increase was observed in the second-wave DF. The diameter of the first-wave DF on Days 8 and 9 was larger than in that of the second-wave DF (Fig. 2). Blood flow in the follicular wall (BFA, blood flow area; BF%, the percentage of the follicular circumference with blood flow signals) was greater in the first-wave DF than in the second-wave DF on Day 9 (Fig. 3). Plasma E2 concentration was higher on Day 8 (P < 0.01) in the first-wave (7.5 ± 0.9 pg/ml) than in the second-wave DF (4.4 ± 0.5 pg/ml).

Fig. 2.

Comparative changes of mean diameter of the first-wave DF and the second-wave DF between Day 3 to Day 9. Day 0 = day of follicular wave emergence. The asterisk denotes difference between each point, * P < 0.05. Values are mean ± SEM of each point. Modified from [21].

Fig. 3.

Comparative changes of (A) blood flow area (BFA), (B) The percentage of follicle circumference with blood flow signals (BF%) at Day 6, 8 and 9 in the first-wave DF and the second-wave DF. Cows were treated with PGF2α and GnRH at Day 6 and Day 8, respectively. Day 0 = day of follicular wave emergence. The asterisk denotes difference between each point, * P < 0.05, ** P < 0.01. Values are mean ± SEM of each point. Modified from [21].

Luteinizing hormone (LH) is necessary for the development of the DF [16]. A higher LH pulse frequency has been observed in cattle with a basal concentration of P4 [22]. Because plasma P4 concentration is lower during the Days 0 to 6 in the first-wave than in the second-wave [21], the frequency of the LH pulse may have been higher in the first-wave than in the second-wave during the development of the DF, and this endocrine condition may lead to the larger size of the DF in the first-wave relative to that of the second wave.

The greater BF%, which represents the degree of distribution of blood flow signals in the follicular wall, in the first-wave DF on Day 9 could indicate that the blood vessels in the follicular wall were more widely distributed than that in the second-wave DF. Therefore, it is likely that the greater blood flow area in the first-wave DF might be caused by greater vascularity in the follicular wall. However, we could not clarify the reason for higher plasma E2 concentration in the first-wave DF than in the second-wave DF from these results.

Follicular Fluid E2 and A4 Concentrations and Amount of mRNA Expression in the Granulosa Cells

To elucidate the reason for higher plasma E2 concentration in the first-wave DF compared with that of the second-wave DF and evaluate the features of the first-wave DF in more detail, aspiration of follicular fluid and collection of granulosa cells were performed from DF during estrus (Day 8) and the preovulatory period (Day 9) in non-lactating Holstein cows [21, 23]. The experimental protocol was same as that in the experiment described above; therefore, the DF at Day 8 was two days after the PGF2α treatment, before the LH surge, and the DF at Day 9 was one day after the GnRH treatment, after the LH surge.

Follicular fluid E2 and A4 concentrations were higher in the first-wave than in the second-wave DF (Table 1). Amount of LHr mRNA expression on Day 8 was higher in the first-wave DF than in the second-wave DF; however, CYP19A1 mRNA expression did not differ between the first and second wave (Fig. 4). On Day 9, amounts of VEGF120, FGF-2 and StAR tended to be higher in the first-wave DF than in the second-wave DF. VEGF164, P450-scc, and 3β-HSD were higher in the first-wave DF than in the second-wave DF (Fig. 5).

Table 1.

The 17β-estradiol (E2) and androstenedione (A4) concentrations in follicular fluid on Day 8 a

	First-wave DF b	Second-wave DF	P-value
E2 (ng/ml)	1942.4 ± 247.9	1169.3 ± 90.9	P < 0.05
A4 (ng/ml)	161.8 ± 26.2	104.6 ± 12.7	P < 0.05

Modified from [21]. Values are means ± SEM. a Day 8 = the day of gonadotropin-releasing hormone (GnRH) treatment. b DF = dominant follicle.

Fig. 4.

LHr and CYP19A1 mRNA amounts of granulosa cells of DF on Day 8. In each figure, the black bar indicates the first-wave DF and the white bar indicates the second-wave DF. Day 0 = day of follicular wave emergence. Different letters indicated P < 0.05. Values are shown as mean ± SEM. Modified from [21].

Fig. 5.

VEGF120, VEGF164,FGF-2, StAR, P450-scc and 3β-HSD mRNA amounts of granulosa cells of DF on Day 9. In each figure, the black bar indicates the first-wave DF and the white bar indicates the second-wave DF. Day 0 = day of follicular wave emergence. Significant differences are indicated by letters; A, B P < 0.1, a, b P < 0.05. Values are shown as mean ± SEM. Modified from [23].

The limiting factor for E2 synthesis in the follicle is the production of A4 in the theca cells rather than the P450 aromatase activity [14]. A4 is produced in theca cells and transported to granulosa cells as an E2 precursor [24]. Wolfenson et al. [18] reported that the productions of A4 and P4 by cultured theca cells were greater in the first-wave DF compared with those of the second-wave DF. A greater concentration of A4 in follicular fluid was observed in the first-wave DF. Consequently, the main reason for the higher E2 concentration in follicular fluid of the first-wave DF may be the higher production of A4 in theca cells; therefore, plasma E2 level is higher on Day 8 in the first-wave.

Luteinizing hormone induces gene expression of LHr in the granulosa cells of cattle [25]. The frequency of the LH pulse may be greater in the first-wave than in the second-wave during the development of the DF because of a lower concentration of P4 [21]. A higher LH pulse frequency might lead to higher LHr mRNA expression. It was presumed that the first-wave DF was more responsive to the LH surge, which may lead to higher mRNA expressions of VEGF120, FGF-2, VEGF164, StAR, P450-scc, and 3β-HSD in the granulosa cells.

These results indicated that the growth, blood flow supply, and steroidogenesis were more active in the first-wave DF than in the second-wave DF.

Growth, Blood Flow, and Plasma P4 Concentration in the CL Formed after Ovulation of the First-wave DF

Amounts of angiogenic and steroidogenic factors were greater in the first-wave DF than in the second-wave DF. Therefore, the luteinization process may be more active during the first-wave DF after the LH surge. It was hypothesized that the CL that formed after ovulation of the first-wave DF has a greater size and greater steroidogenic capacity; therefore, we investigated the growth, blood flow, and plasma P4 concentration in the CL formed after ovulation of the first-wave DF in comparison with that of the second-wave.

The cross-sectional area of the CL, blood flow area in the CL, and plasma P4 concentration were higher in the CL formed after ovulation of the first-wave DF than after the second-wave DF (Fig. 6).

Fig. 6.

Comparative changes of cross-sectional and blood flow areas of corpus luteum (CL), and blood progesterone (P4) levels in first or second-wave CL. X axis showed days from ovulation of the first- or second-wave DF. The asterisk denotes difference between each point of same day, * P < 0.05. Values are mean ± SEM of each point. Modified from [23].

In a previous study, the local administration of a VEGF antagonist (soluble VEGF receptor) into the preovulatory follicle impaired the subsequent structure and function of the CL [26]. Furthermore, treatment with an FGFR1 inhibitor using bovine luteal cells caused a maximal reduction in the total area of the endothelial cell networks and reduced the total number of branch points and degree of branching per endothelial cell island [27]. Expression levels of VEGF120, VEGF164, and FGF-2 were higher in granulosa cells of the first-wave DF than the second-wave DF. This may lead to higher blood flow area and size of the CL formed after ovulation of the first-wave DF.

Progesterone is synthesized in luteal cells by several steroidogenic enzymes such as StAR, P450-scc and 3β-HSD [28]. StAR, P450-scc, and 3β-HSD mRNA in granulosa cells were higher in the first-wave DF than the second-wave DF; therefore, it was expected that the steroidogenic capacity of the luteal cells would be greater in the first-wave CL than that in the second-wave CL. In addition, size of the first-wave CL was larger than that of the second-wave CL. Taking these findings together, greater steroidogenesis and size in CL formed after ovulation of the first-wave DF may lead to a greater plasma concentration of P4.

Thus, ovulation of the preovulatory follicle of the first-wave DF leads to the formation of a more active CL than that of the second-wave DF.

Conception Rates between the First-wave DFs that are Ipsilateral and Contralateral to the CL in the Ovaries

The dynamics of DF growth and the hormonal milieu during first-wave development have been well studied [13]. However, the influence of the first-wave DF on fertility (conception rate) in cattle remains unclear. Furthermore, the first-wave DF develops contralateral or ipsilateral to the CL (Fig. 7). In a previous study, the relative locations of the first-wave DF and CL were determined, and the number of follicular waves with the DF and CL ipsilateral or contralateral to the ovaries did not vary during the first-wave [29]. However, the locational effects of the first-wave DF that is contralateral or ipsilateral to the CL on fertility remains unevaluated. Therefore, we compared conception rates between the first-wave DF that were ipsilateral and contralateral to the CL in the ovaries of lactating dairy cows and dairy heifers [30].

Fig. 7.

Schematic figure of the locational relationship between the first-wave dominant follicle (DF) and corpus luteum (CL).

A total of 238 artificial inseminations (AIs) in lactating dairy cows [average postpartum day of AI, 119.8 ± 5.2; average parity, 2.2 ± 0.1; average milk production, 33.9 ± 0.5 kg/day; average body condition score (BCS), 2.91 ± 0.02; average live weight, 637.4 ± 4.6 kg; mean ± SEM] and 112 AIs in dairy heifers (average age, 14.2 ± 0.3 months; average live weight, 440.6 ± 5.0 kg) were analyzed. These replicates underwent regular estrous cycles and were clinically healthy during the breeding period. If the lactating dairy cow experienced reproductive or metabolic diseases, we excluded it from the study.

The location of the first-wave DF in the ovary was confirmed at that time to be ipsilateral (ipsilateral group, IG) or contralateral (contralateral group, CG) to the CL during Day 5 to 9 after AI. Conception rates were 54.0% in all cattle, 48.9% in lactating dairy cows and 58.9% in dairy heifers.

Conception rates were lower in IG than in CG of both of lactating dairy cows and dairy heifers (lactating dairy cows, 38.0% vs. 67.0%; dairy heifers, 45.5% vs. 75.4%; IG vs. CG: Fig. 8.). Additionally, we analyzed the effect of season, days in milk at AI, milk production, body condition score, parity, or live weight on conception rate, but these factors did not affect conception rates in either group in this study.

Fig. 8.

Conception rates in ipsilateral group (IG) and contralateral group (CG). The asterisk denotes difference between each point of same day, * P < 0.01. Modified from [30].

The endometrium of the uterine horn in cattle, which is located on the same side of the ovary as the CL, has a higher P4 concentration compared with that of the endometrium in the opposite uterine horn [31, 32]. In addition, the concentration of E2 in the oviduct ipsilateral to the preovulatory follicle is higher than that of the one contralateral to the preovulatory follicle in bovine [33]. These results indicate that there is a strong local interaction between the uterine horn and the same side ovary. The first-wave DF produces and secretes E2 during follicular development in cows [20]. Therefore, it is hypothesized that E2 secreted from the first-wave DF may locally affect the same side of the uterine horn and/or oviduct and affect the function of the reproductive tract, which was associated with decreased fertility in IG. However, the mechanism of lower fertility when the first-wave DF is ipsilateral to the CL remains unclear. We require further research to evaluate the steroid hormones, such as P4 and E2, concentrations and mRNA expression of steroid hormone receptors in oviduct and the endometrium of IG and CG.

Consequently, the locational relationship of the first-wave DF and CL affects the conception rate after AI, in particular, ipsilateral location relative to the CL in the ovary was associated with reduced conception rates in both lactating dairy cows and dairy heifers.

Effect of hCG Treatment 5 Days after AI on the Conception Rate in Ipsilateral and Contralateral Situations

Human chorionic gonadotropin (hCG) has a luteinizing hormone-like effect in cattle [34]. During the early luteal phase, hCG induces ovulation of the first wave DF and the formation of an accessory CL, with a subsequent increase in plasma P4 concentrations in cattle [35]. On the basis of the increased plasma P4 concentrations, several trials of hCG treatment after AI in the early luteal phase of lactating dairy cows have been performed to increase conception rates [36]. However, the effects of hCG administration on fertility are not consistent between studies [37, 38]. From the results of previous chapter, the development of the first-wave DF in the ovary ipsilateral to the CL was associated with reduced conception rates in lactating dairy cows. On this basis, removing the first-wave DF that develops ipsilateral to the CL using hCG treatment could eliminate the detrimental effects on fertility, and thereby, possibly increase conception rates [39].

We performed 599 AIs in lactating dairy cows in four dairy farms (postpartum day of AI, 125.4 ± 62.6; parity, 2.4 ± 1.5; means ± SD). Cows underwent regular estrous cycles and were clinically healthy during the breeding period. Cows that experienced reproductive or metabolic diseases were excluded. They were randomly assigned to either the non-treatment group or hCG treatment group 5 days after AI. The cows in the non-treatment group (n = 363) were not administrated hCG after AI, whereas those in the hCG treatment group (n = 214) were intramuscularly administrated 1500 IU hCG 5 days after AI. In addition, the location of the first-wave DF in the ovary was confirmed to be either ipsilateral (IG: non-treatment, n = 220; hCG treatment, n = 128) or contralateral (CG: non-treatment, n = 143; hCG treatment, n = 86) to the CL.

Conception rate increased in the hCG treatment group with the IG (40.6%) more than in the non-treatment group with the IG (21.4%); however, there was no difference in the non-treatment (51.7%) and hCG treatment (43.0%) groups with the CG (Fig. 9). Parity, farm, days in milk at AI, interaction between the farm and hCG treatment, and interaction between the farm and location of the first-wave DF and CL did not affect conception rate.

Fig. 9.

Effect of hCG treatment 5 days after AI on the conception rate in ipsilateral group (IG) and contralateral group (CG). The asterisk denotes difference between each point of same day, * P < 0.01.

The physiological mechanisms of increasing conception rate by hCG treatment in IG were not completely clarified in this study. In a previous study, lactating dairy cows that were diagnosed as pregnant have a higher P4 concentration from 6 to 8 days after AI compared to that of non-pregnant cows [40]. However, plasma P4 concentration was not different between IG and CG in lactating dairy cows (unpublished data). Thus, the higher conception rate in CG compared to IG might be not caused by a greater plasma P4 concentration in CG than IG. It is possible that E2 secreted from the first-wave DF might have locally affected the same side of the uterine horn or oviduct and affected the function of the reproductive tract. Because ovulation of the first-wave DF in the IG condition with hCG treatment could eliminate the detrimental effects on fertility, the conception rate increased in IG. On the contrary, it has been reported that the P4 concentration in the endometrial tissue is higher in the ipsilateral location of the CL in the ovary [31, 32]. Therefore, because hCG treatment with the IG condition had an accessory CL and original CL in the same side of the ovary, the P4 concentration in the oviduct and uterine horn under the ipsilateral condition of the CL might be higher and have a positive effect on fertility compared with the IG condition with no hCG treatment.

However, we could not evaluate the purely local negative effect of the first-wave DF on fertility. Further research is warranted to verify the locational effect of the first-wave DF on fertility following follicle aspiration after AI.

From the present study of lactating dairy cows, it was shown that hCG treatment 5 days after AI had a beneficial effect on fertility only when the first-wave DF developed ipsilateral to the CL in the ovary, and not when the first-wave DF developed contralaterally.

Conclusion

We showed that 1) compared to the second-wave DF, the first-wave DF had greater size, blood flow in the follicular wall, plasma E2 concentration, follicular fluid E2 and A4 and mRNA expression (LHr, VEGF, FGF-2, StAR, P450-scc, 3β-HSD) when estrus was induced by hormonal treatment, 2) the CL formed after ovulation was greater in terms of size, blood flow, and plasma P4 concentration in the first-wave DF than in the second-wave DF, 3) the first-wave DF located ipsilateral to the CL in the ovary was associated with reduced conception rates, and 4) hCG treatment 5 days after AI had a beneficial effect on fertility only when the first-wave DF developed ipsilateral to the CL in the ovary, and not when the first-wave DF developed contralaterally. Further studies are needed to clarify the physiological mechanism of these phenomena. We suggest that progress in such research would provide new perspectives on the reproductive physiology of cattle and the development of clinical applications for improvement of reproductive performance in the cattle industry.