**4. Developmental stages of ovarian follicles**

Folliculogenesis, the complex biological process of forming ovulatory follicles among the cohort of growing primordial follicles on the ovaries produced by

### *The Incidence of Ovulation and Detection of Genes Associated with Ovulation and Twinning… DOI: http://dx.doi.org/10.5772/intechopen.99936*

female animals throughout their lifetimes, causes changes in ovarian morphological characteristics during the typical estrus cycle, an essential aspect of female reproduction [32]. Cattle are a monovular species that can produce several hundred thousand primordial follicles at the onset of puberty, depending on their physiological mechanism. However, practically less than 1% of these follicles will grow and be ovulatory in the late stages of development due to atresia. Selecting a single dominant follicle among many growing primordial follicles is an essential step in livestock reproductive technology. Therefore, any intervention or malfunction in this process can lead to infertility or multiple ovulation in females. The current follicle selection process focuses on the role of follicle growth and selection of the dominant follicle regulated by LH, FSH, and insulin-like growth factor family (IGF) hormone mechanisms [33].

Many studies on the growth stages and developmental processes of follicles in animals have also presented different models. As one of the most notable models, Rajakoski proposed the developmental stages of antral ovarian follicular growth in cattle occur in a wave-like pattern [34]. Many researchers reported that each cycle usually involves two or three waves. As a result of applying transrectal ultrasonography technology, the concept of follicular waves known to that day has been re-investigated and facilitated the understanding of the pattern of follicular waves during the estrus cycle [35, 36]. Therefore, ultrasonography technology has provided more detailed information about the follicular developmental stage and the follicular wave dynamics. In addition, monitoring the growth and development patterns of follicles has enabled us to make more detailed observations about follicle selection and understand how it relates to the endocrine secretion mechanism during the maturation of follicles [36, 37].

First, Pierson and Ginther observed individual follicular development during the cycle using this technology [37]. Later, various intensive studies were carried out to investigate these developmental stages of animal husbandry, especially in sheep and cattle breeds [38, 39]. The follicular wave pattern in ruminants is typically two or three follicular waves per cycle in cattle; incredibly primitive and very fertile dairy cows usually have two wave cycles, while nulliparous dairy heifers aged 2–2.5 years have three-wave cycles [40]. However, it is three to six waves per cycle in sheep [41]. Studies were proving that the developmental processes of follicles within a follicular wave are highly variable among waves. After puberty, all primordial follicles have an equal chance of becoming mature follicles. Primary follicular wave is characterized as the synchronous growth of a group of small antral follicles. One of them is eventually selected to be dominant and thereby becoming ovulatory among the group of follicles within each follicular wave. But all other remaining "subordinate" follicles of the ovulation wave will regress and degenerate during the typical estrus cycle [42, 43]. The dominant or mature follicle of the wave is typically the largest in diameter. Still, the subordinate follicles belong to the same group of follicles which the dominant follicle comes from [40].

Traditionally, in cattle, the day when a follicular wave can be first detected determines the day when the first observation of the dominant follicle can be made retrospectively [44]. A first dominant follicular wave emerges when the follicles are 4–5 mm in diameter at approximately the day of ovulation [42]. Subsequently, a second ovulatory wave can be detected about 9 or 10 days later [45]. The main event that causes single ovulation to occur in cattle is called follicle selection. Diameter deviation occurs approximately 2 to 3 days after the emergence of the follicular wave in the selection of follicles in the morphological process. Thus, while the future dominant follicle grows continuously, the growth rate of the lower follicles slows down, and then their growth is stopped entirely, and they undergo degeneration. Although this deviation varies among individual animals, it is widely accepted

as it has been observed in this range in many studies using both Bos taurus and Bos indicus breeds [46–48]. However, the high progesterone concentration prevents the first dominant follicle from maturing, as the corpus luteum has not regressed yet. Thus, the first dominant follicle cannot be functional and ovulatory. Subsequently, a second ovulatory wave can be observed. The dominant follicle from this wave can keep on growing and ovulating during the corpora lutea (CL) regression. In addition, a third source of the ovulatory follicle becomes apparent on day 16 after ovulation in some cattle breeds due to the regression of the second dominant follicle during luteolysis. Even if each wave involves simultaneous emergence of a cohort of follicles, usually one of them, sometimes two, become dominant follicle(s), and all of the others eventually become subordinates. A single oocyte is released from the dominant follicle due to either naturally occurring or artificially induced ovulation. On the other hand, subordinate follicles begin to regress right after a short growing phase [44, 45].

It was noticed from individual to individual that the follicle size at ovulation was quite different. Dairy heifers showing two-wave cycles have a follicle at a diameter of 16.5 mm in ovulation. However, follicle size is smaller in heifers (13.9 mm) with three-wave cycles [44]. Similarly, the size of ovulation follicles has been reported as 14.8 mm in Holstein heifers. However, the follicle size observed for lactating dairy cows was slightly larger and was found to be 17.4 mm [49]. In many studies of follicular diameter deviation, both the future dominant follicle and the most significant lower follicle were more prominent in Bos taurus. However, diameter deviation occurred at similar times after wave emergence in Bos taurus and Bos indicus. Bos indicus has a smaller follicle size when the deviation in the follicle diameter cannot be fully revealed. Nevertheless, the results of the studies support the idea that the future dominant follicle generally has a size advantage over the largest subordinate one [46, 48, 50].

Reproductive biotechnology has recently emerged as a powerful tool to improve livestock productivity and reproductive performance. Therefore, these modern reproductive technologies have started to be used instead of conventional classical techniques in many reproductive-based studies recently. Progress in our understanding of follicle development and selection has sparked the development of synchronization protocols for fixed-time artificial insemination (AI), in addition to the applications of other cutting-edge reproductive technologies such as in-vitro fertilization (IVF), embryo culturing and transfer (ET), cloning, estrus synchronization, transgenesis, and much more new emerging reproductive biotechnologies [51, 52]. As a result, these developments in terms of sustainable livestock productivity are important for optimal follicle growth and making the right choices to increase reproductive efficiency in livestock species.

## **5. The incidence of multiple ovulations**

Cattle are a uniparous species that means females usually produce only one progeny per conception due to the single dominant follicle in each ovulatory cycle. Alterations in follicle selection can lead to codominant follicles and multiple ovulations, which are the basis for multiple births in cattle and sheep [53]. In rare cases, the synchronous emergence of two follicles as a physiological pattern, albeit in a monovular type, is altered so that the follicle selection mechanism allows both to be selected as the dominant follicle among several follicles in the follicular wave. The ease of evaluating follicular events by trans-rectal ultrasonography and the accuracy of the data obtained from these studies have allowed cows to be widely used as an ideal research model in follicular studies in ruminants and humans [54, 55].

### *The Incidence of Ovulation and Detection of Genes Associated with Ovulation and Twinning… DOI: http://dx.doi.org/10.5772/intechopen.99936*

Ultimately two oocytes are released from codominant follicles at the end of ovulation due to either natural stimulation or artificial inducements. In the development of codominant follicles, deviations occur in the diameter of the follicles when the largest follicle and the second-largest follicle are close to 8.5 mm. The third-largest follicle has a low growth performance, and the deviation in 2nd largest follicle may occur 36–50 h after the deviation of the first follicle [56]. Ovulation of two future dominant follicles occurs either from the same ovary simultaneously, or each follicle consists of a separate ovary [57]. In a study, synchronous production of two oocytes from different follicles was observed due to the evidence of two corpus luteum (CL) on the ovaries of cattle [58]. Also, research about follicular development during the estrous cycle in twin-calving cows indicated that double and triple ovulations coincide from different ovulatory follicles of the same follicular wave rather than ovulation of single mature follicles from two consecutive waves [59]. In addition, the authors noted that the cysts in the ovary and lack of CL possibly increased the incidence of double ovulation during pregnancy [60]. Therefore, as more than one follicle deviates and becomes dominant, the chance will be increased for ovulation of more follicles simultaneously. After all, twins, triplets, or overall multiple births in rare circumstances will become a reality if all subsequent events commonly occur for both oocytes from fertilization to parturition.

The natural incidence of twin or triplets birth in cattle is mainly due to multiple ovulations that have been summarized in many studies, even if the results are inconsistent [61]. While the multiple births five decades ago is around 1–5% depending on breed, genetics, parity, and other environmental conditions [62], this rate has increased up to 10–22% in lactating dairy cows today. There have been many studies conducted about regulating multiple birth rates in cattle by selecting genetically favorable animals [63], utilizing hormonal treatments [64], or utilizing embryo transfer techniques [65]. One of the reasons affecting ovulation rates is low progesterone secretion in older cows, which might be the main reason for the increase in circulating LH level and eventually causes enhance ovulation numbers as progesterone has a suppressive effect on LH release. In addition, growth hormone and nutritional treatments greatly influence a multi-ovulation response of an individual [66]. Also, the ovulations of two follicles simultaneously caused to increase in days of milk among pregnant animals [60]. In a recent study, the incidence of multiple ovulation rates in early lactating animals was 14.1%, but they did not significantly affect various reproductive outcomes of cattle [67]. Although the underlying mechanisms of multiple ovulation have been studied extensively, the dynamics of the entire mechanism are still not fully explained.

Monozygotic twins are genetically and physically identical since they are formed from one fertilized egg, splitting into two identical halves during early embryonic developmental stages. Thereby both individuals are always the same sex. In the case of dizygotic or fraternal twins, two different sperm fertilize two completely different ova simultaneously. Thus the successful result of ovulation and fertilization of two oocytes will be dizygotic twins. Dizygotic twins are not identical genetically or phenotypically as monozygotic twins are. They are not necessarily the same sex as opposed to monozygotic twins. They can also be similar or different from siblings born from the same parents during different gestations [68].

Twin or triplets birth is an unavoidable issue in dairy and beef cattle production systems which negatively affects the health, production, reproduction and overall decreases the productive life span of animals [69]. The study reports that the calf survival incidence from twin- and triplet-producing animals were relatively low, around 44% depending on the breed composition [70]. In a recent study of the economic analysis of multiple births, the economic loss to the livestock breeder from each twin calving was estimated at between \$59 and \$161 in cow-calf

production systems [71], even if twin calving could reduce substantially beef meat production costs owing to more calf growth at weaning [69]. Thus twin or triplet calving causes to lessen overall cow reproductive efficiency, productivity, and thus the profitability of enterprises. In conclusion, a complete understanding of the complex process of follicular growth during the estrus cycle and the development of oocytes will undoubtedly improve the knowledge to maximize and control the efficiency of reproduction in livestock species, especially the existence of dizygotic twinning since the fertilization of more than one oocyte after ovulation will be the main reason of multiple births.
