Application of Ultrasonography in Bovine Reproduction

Abstract

With the introduction of improved concepts in breeding strategies (aiming at enhancing the production potential of the animals), an increase in infertility is obviously evident. It has posed new challenges among the farmers as well as the physicians. These challenges in bovine breeding systems warranted introduction of new user-friendly diagnostic technologies. Ultrasonography is a simple and potent technology that can strengthen the diagnostic range in the field conditions. With the help of ultrasound technology, it is possible to assess the entire reproductive system in a non-invasive manner. Reproductive ultrasonography is a dynamic introduction in the veterinary research and clinical investigation. Since 1980s, the ultrasonographic investigation has revolutionized the field of bovine reproduction. The technology has opened new insights in the better understanding of basic reproductive physiology as well as diagnosis of occult pathological conditions. The basic physical principles of ultrasonography, the proper use of the instrument’s controls, the anatomical positions of the structures to be scanned, the interactions between the ultrasound wave and the organ tissue (the basis for obtaining quality images) and the interpretation of the obtained images appropriately applications of ultrasonographic investigation in reproductive physiology and pathology are discussed in detail in this chapter.

Reproductive ultrasonography in bovines can be performed using sectorial or linear probe. Sectorial probe (trans-vaginal) doesn’t require a large surface of contact, and it scans a greater overall surface and the latter (transrectal) provides good resolution for tissues located close to the probe. Transrectal linear-array transducer is easy to handle, animal friendly and can be used for more detailed study of utero-ovarian structures than the trans-vaginal probe. Hence, linear-array transducers are most commonly used in cattle for reproductive ultrasonography.

Depth of tissue penetration of sound waves and image resolution is dependent upon and inversely related to the frequency of the transducer. Thus, a 5.0 MHz transducer results in greater tissue penetration and lesser image detail, whereas a 7.5 MHz transducer results in lesser tissue penetration and greater image detail. The anatomical association of rectum and genitalia warrants transducers of 5–7.5 MHz frequency for the investigation of utero-ovarian structures.

The basics of ultrasonographic imaging depend on the echogenic or echoic nature of the particular organ or tissue (Fig. 2.1).

• An ‘echogenic’ or ‘echoic’ structure, such as bone and tissue, reflects the majority of sound waves back to the probe and thus appears from white to different shades of grey on the screen. Hypoechogenic and hyperechogenic refer to a decrease or an increase in relative echogenicity in comparison with the surrounding tissue, while isoechogenic refers to similar echogenicity with the surrounding tissue.

• An ‘anechogenic’ or ‘anechoic’ structure, such as fluid, does not reflect the sound waves, and the image appears black on the screen.

Fig. 2.1

Ultrasonographic imaging depends on the echogenic nature of the tissue

2.1 Ultrasonographic Imaging of Ovarian Structures

Transrectal ultrasonographic imaging is an effective tool for characterizing ovarian structures (ovarian parenchyma, follicle and corpus luteum) and tubular genital tract along with adnexa. It is non-invasive and permits repeated examinations of the utero-ovarian structures. Follicles that are below the ovarian surface and embedded CL, whose palpation by regular gynaeco-clinical examination is ambiguous, can be detected with precision using the ultrasonographic aids.

2.1.1 Imaging of Follicle

The most distinguishable ovarian structures are antral follicles. Antral follicles are fluid-filled structures and hence allow the ultrasound waves to pass through (i.e., anechoic or anechogenic) and are displayed as black images on the screen surrounded by hyper/hypoechogenic ovarian tissue (Fig. 2.2). Each ovary should be scanned and imaged in more than one plane to ensure that all measurable follicles were detected.

Fig. 2.2

Ultrasonographic imaging of follicle and CL. (a) Dominant follicle, (b) Corpus luteum, (c) Cystic corpora lutea

2.1.2 Imaging of Corpus Luteum

Corpus luteum (CL) appears as distinctly echogenic area within the ovarian stroma. The ovarian stroma and CL contain varying degrees of granular cells and result in a grey image on the screen. Usually CL appears as a solid tissue mass, but some CL may contain fluid-filled cavity (cystic corpora lutea) and the cavities range from <2 to >10 mm in diameter (Fig. 2.2). Embedded CL, which is often difficult to be palpated by per-rectal examination, can be documented clearly with the ultrasound technology.

2.1.3 Imaging of Uterus

In a non-pregnant cycling cow, the uterine tissue normally appears as a partially echogenic structure on the screen. Since the uterus is comprised of soft tissue, it absorbs a portion of the ultrasound waves and reflects a portion of the waves. In this way, we can identify the uterus as a grey structure either in the cross section or sagittal section view on the screen (Fig. 2.3).

Fig. 2.3

Ultrasonographic imaging of non-pregnant uterine horns—cross section and sagittal section view

2.1.4 Biometry of the Utero-Ovarian Structures

The inbuilt electronic caliper of the ultrasound equipment can be utilized to measure the length and width of the ovarian structures, and the diameter was determined by taking the mean of these two values (Savio et al. 1988). Based on the ultrasonographic studies in crossbred cattle over years, certain biometrical standards were arrived for clinical assessment of the structures, as follows.

• 1. Follicles with diameter of >8.5 mm are considered as dominant follicle (DF).

• 2. CL with diameter of >15.0 mm is considered as mature and active.

• 3. CL with diameter of <15.0 mm is considered as regressed structure.

Likewise, the thickness of uterine endometrium, embryonic/foetal dimensions, etc. can be measured in a linear fashion using the electronic caliper in the ultrasound equipment.

2.2 Ultrasonographic Characterization of Folliculogenesis and Luteal Development

In cattle, ovulatory follicles do not develop randomly, but their development occurs in a regular fashion. For better understanding of ovarian physiology, various investigative techniques have been used to characterize the follicular development in cattle including the histological evaluation of ovaries, the correlation of follicular inventories with measurement of steroids in follicular fluid and laparoscopic monitoring of individual follicles by marking them with dyes. The studies and advancements that have led to our current understanding regarding patterns of follicular development are listed below in chronological order:

1960 The two-wave concept for follicular growth during the bovine oestrous cycle was proposed (Rajakoski 1960).

1972 and 1981 The lifespan and fate of individual follicles during the oestrous cycle of heifers were directly examined (Dufour et al. 1972; Matton et al. 1981).

1982–1983 Growth and differentiation of oestrogen-active and oestrogen-inactive follicles during the oestrous cycle were distinguished (Ireland and Roche 1982, 1983).

Although these earlier studies provided important information, they precluded the sequential monitoring of individual follicles over a long duration, which led to conflicting ideas about follicular dynamics in cattle. With the advent of real-time ultrasonography, it became possible to visualize the various reproductive events in large animal species, especially cattle, over a prolonged period of time and without any interruption of normal physiological events. The modern era of study of follicular dynamics in cattle began with the classic publication by Pierson and Ginther in the year 1984.

1984 Ultrasound to monitor sizes of follicles during the oestrous cycle of heifers was used (Pierson and Ginther 1984).

1987 The concept of DFs, as observed in primates, was applied to cattle, and the three-wave hypothesis for development of DFs during the oestrous cycle was proposed (Ireland 1987).

1988 Ultrasound analysis and ovarian maps to track growth and atresia of individual follicles throughout the oestrous cycle of heifers were used (Savio et al. 1988; Sirois and Fortune 1988).

Since 1984, the application of real-time ultrasonography in monitoring the utero-ovarian function took off in a jet propulsion and has advanced the understanding of follicular and luteal dynamics during the cycle.

2.2.1 Ovarian Follicular Dynamics

Based on the preliminary ultrasonographic documentation, Pierson and Ginther (1984) recorded that there was (1) growth of a large follicle to an ostensibly ovulatory size followed by regression at approximately mid-cycle, (2) selective accelerated growth of the follicle destined to ovulate approximately 3 days before ovulation and (3) a few days before ovulation regression of the larger follicles that were not destined to ovulate.

Further investigations have proved that folliculogenesis operates on a wave-like fashion at 7–8 day intervals throughout the oestrous cycle in B. taurus (Ginther et al. 1989a, b; Pursley et al. 1993), B. indicus (Viana et al. 2000; Gaur and Purohit 2007) and B. taurus x B. indicus crossbred (Satheshkumar et al. 2008a, 2011, 2012a; Satheshkumar 2015a) cattle.

Follicular wave is a sequence of organized events consisting of the synchronous growth of small (4–5 mm) antral follicles (recruitment), followed by the selection, deviation and growth of one dominant follicle (DF) which achieves the largest diameter and suppresses the growth of the subordinate follicles. In the absence of luteal regression, the DF eventually regresses (becomes atretic) and a new follicular wave begins in a day or two. The DF regulates the growth of the subordinate follicles because the appearance of the next wave is accelerated if the DF is ablated, and delayed if the lifespan of the DF is prolonged. These sequence of events comprising of recruitment, selection, deviation, growth, dominance and regression of a DF is known as a follicular wave. During bovine oestrous cycles, two or three successive waves emerge, on average, on the day of ovulation (day 0) and day 10 for two-wave cycles, and on days 0, 9 and 16 for three-wave cycles (Duggavathi et al. 2003). Thus, there two or three successive DFs develop during an oestrous cycle, and the last of these ovulates with the advantage of luteolysis (Fig. 2.4).

Fig. 2.4

Different follicular wave patterns during the oestrous cycle

Two and three follicular wave patterns are common among the Indian crossbred cattle but their incidence during an oestrous cycle is highly variable and unpredictable (Satheshkumar et al. 2008a, b; Satheshkumar 2015a, b). These variations in follicular wave patterns are one of the major contributory factors of fertility disturbances among the dairy cattle population. It was reported that oestrous cycles with three follicular waves have better fertility than those with two follicular waves (Townson et al. 2002). Various factors govern the follicular wave pattern during an oestrous cycle like the plane of nutrition (Murphy et al. 1991), circulating endocrine status (Adams et al. 1992), season (Satheshkumar et al. 2015), luteal status (Satheshkumar 2020), etc.. However, the basic ultrasonographic studies have given the concept for inducing the occurrence of three follicular wave cycles by hormonal intervention (Satheshkumar et al. 2012a, b). When GnRH analogue (Buserelin acetate–10 μg) was administered on Day 5 or 6 of the cycle, the DF of first follicular wave ovulated and a synchronized homogenous group of follicles emerged after 2 days. Because of early emergence of second follicular wave and high progesterone (P₄) concentration in the mid-cycle due to accessory CL formation, the DF of second follicular wave will regress and eventually the third wave arise (Fig. 2.5). Thus, positive manipulation of follicular wave emergence is made possible because of better understanding of follicular physiology.

Fig. 2.5

GnRH induced occurrence of three follicular waves

2.2.2 Ovarian Luteal Dynamics

Fertility of any mammalian species is closely related to their luteal activity. In cattle, CL is the prime source for P₄ synthesis and hence its sustained function needs to be secured in order to support pre-implantation embryonic development and to maintain the pregnancy (Roberts 1971; Grana-Baumgartner et al. 2020). Ginther et al. (1989a, b) reported that the time of luteal regression is one of the factors that determine the number of follicular waves during a cycle. The pattern of luteal development was sequentially drafted using the transrectal ultrasonography in crossbred cattle (Satheshkumar 2020). In general, the CL grew progressively until it attained the maximum diameter (21.7 ± 0.4 mm) during the mid of the cycle (mean day 9.5 ± 0.4) and remained fluctuating around that size for a certain period (period of luteal dominance; 5.7 ± 0.6 days) before it started regressing constantly (from Day 15.2 ± 0.8) towards the end of the cycle.

The variations in luteal development characteristics were observed in oestrous cycles with two follicular waves and three follicular waves (Fig. 2.6). The maximum diameter of CL was significantly larger in three-wave cycles than that of two-wave cycles. In cycles with three follicular waves, the CL reached the maximum diameter significantly earlier, remained dominant for a significantly longer duration and started to regress non-significantly later when compared with cycles of two follicular waves. The early attainment of maximum diameter and late regression of CL during a three-follicular wave cycle assured a significantly increased period of luteal dominance than the two-follicular wave cycles. Thus, it could be concluded that the period of luteal dominance and the associated luteal endocrine activity have a possible control over follicular turnover during spontaneous oestrous cycle in crossbred cows. Taking into consideration of the importance of luteal support on embryonic sustenance and establishment of pregnancy, it is very obvious to associate the luteal insufficiency and prolonged persistence of second wave DF with the poor fertility in cows with two-follicular wave cycles.

Fig. 2.6

Luteal dynamics during two- and three-follicular wave cycles

2.3 Role of Ultrasound in Diagnosing Physiologic Reproductive Events and Its Variations

The reproductive efficiency of an animal is influenced by many extrinsic factors such as nutrition, lactational status, season, breeding practices, etc. However, recent epidemiological studies indicate that intrinsic factors like variations in follicular and luteal development contribute to the fertility disturbances in dairy herds. Ovarian follicles are the vital units which provide proper environment and nourishment for the female gametes, the oocytes and contribute for the emergence of healthy CL. The endocrine status of both these structures provides a protective uterine environment for the establishment of pregnancy. Any deviation in this utero-ovarian chain during the oestrous cycle can directly or indirectly affects the fertility of the animal. In addition to the routine gynaeco-clinical examination, the ultrasonographic investigation provides a confirmatory aid to assess the utero-ovarian characteristics in cattle and to manipulate it positively for enhancing the fertility.

2.3.1 Diagnosis of Oestrum and Its Aberrations

Oestrus is the most precious phase of oestrous cycle and is the gateway for fertility. Efficient oestrus detection is a key factor that determines the success of breeding program in a dairy herd. In spite of progress in the development of oestrus detection aids, errors occur leading to setback in reproductive management of dairy cows. Variations in the duration and intensity of oestrus warrant the need for accurate and continuous monitoring to determine the optimal breeding time. The low accuracy and efficiency of oestrus detection not only increases time from calving to first AI but also increases the average interval between services (Stevenson and Call 1983), thereby limiting the overall pregnancy rate.

In the field conditions, whether all the animals are being inseminated in the actual oestrus? Nebel et al. (1987) reported that insemination of cows not in oestrus was a problem in 30% of the herds and emphasized that errors in detection of oestrus must be considered as a significant cause of low conception rates in problem herds. The behavioural changes, clinical signs, and per-rectal gynaeco-clinical investigation of uterine status were considered to be the important approaches in diagnosing oestrus in cattle, but it is quiet astonishing to know the fact that many animals are being inseminated in the wrong time of the oestrous cycle with these routine diagnostics.

Anoestrus, prolonged oestrus and silent oestrus are the widely discussed aberrations of oestrus expression pattern in cattle, but in the recent years occurrence of ‘mid-cycle oestrus’ (MCO) is found to be emerging as a serious aberration in the crossbred cows (Sood et al. 2009; Satheshkumar et al. 2014a, b; Satheshkumar et al. 2019a, b). The exhibition of oestrus-like signs during the early or mid-luteal phase (Day 6–12) of the cycle is referred as MCO. Apart from external signs like restlessness, vaginal discharge, mounting, etc., routine gynaeco-clinical examination also revealed moderate to good tonicity of uterine horns simulating the oestrus. These factors mislead the farmers and the veterinarians equally in diagnosing the oestrus. Sood et al. (2009), Satheshkumar et al. (2014a) and Satheshkumar (2018a) recorded the incidence of MCO among crossbred cattle as 2.4, 6.2 and 8.8%, respectively. Perusal of these reports revealed that the incidence of MCO is in increasing trend and is also equally limiting the reproductive outcome in cattle.

Luteal deficiency is the major factor contributing to the occurrence of MCO (Satheshkumar 2018a). The increased oestradiol (E₂) concentration secreted by the DFs of first or second follicular wave overrode the P₄ and induced the animal to exhibit oestrus-like signs during the mid-cycle. Animals won’t conceive when inseminations are carried out during MCO because P₄ concentration during diestrum is sufficient enough to prevent the ovulation. Further, there is every likelihood of inducing embryonic mortality by inseminating the animal that would have conceived to previous insemination which was done 7–10 days back. Number of inseminations per conception tends to increase when animals are inseminated repeatedly during MCO, thereby falsely projecting the incidence of repeat breeding condition.

With the application of ultrasonographic imaging technology, animals in proper oestrus are diagnosed with more precision. Before proceeding for insemination based on routine clinical examination, ultrasonographic assessment of DF and CL characteristics provides the exact reproductive status of the animal. How? Biometric assessment revealed that the mean diameters of DF and regressed CL during oestrus were 11.9 ± 1.5 mm and 12.7 ± 1.7 mm, respectively. However, the corresponding values in MCO were 10.9 ± 1.1 mm and 19.3 ± 1.0 mm, respectively. There was no significant difference in the diameter of largest follicle present during oestrus and MCO, but the diameter of CL was significantly larger in MCO (matured CL during diestrum) than oestrus (Satheshkumar 2018a). Hence, based on ultrasonographic examination, a basic thumb rule can be followed before deciding to perform insemination in the animal (Fig. 2.7).

• A combination of a DF (with >8.5 mm diameter) and regressing CL (with <15.0 mm diameter) indicates oestrus. Animal can be inseminated.

• A combination of a DF (with >8.5 mm diameter) and prominent/mature CL (with >15.0 mm diameter) indicates the MCO. Insemination should be avoided.

Fig. 2.7

DF and CL status during oestrus and MCO (a) Oestrus, (b) Mid-cycle oestrus

Assessment of CL size by ultrasonographic examination should be practiced regularly to distinguish between oestrus and MCO, thus inseminations at correct stage of the cycle are ensured.

2.3.2 Diagnosis of Ovulation and Its Disturbances

Ovulation is an important physiological event that determines the fertility of an animal and any disturbance in this event will result in anoestrum, cystic degeneration or repeat breeding syndrome. Normally ovulation occurs 10–12 h after the end of oestrum in cattle. In the recent years, we could observe lot of disturbances in the ovulatory processes, especially delay in the timing of ovulation which is evidenced as prolonged oestrus.

A sequential ultrasonographic study (12 h interval) of the DFs from the onset of oestrus gave a clear indication about the incidence of ovulatory disturbances in crossbred cattle (Satheshkumar 2018b). Ovulation was confirmed when the DF was no longer seen in the same location on subsequent examination (Kim and Kim 2007). The follicular size increased non-significantly until the last examination prior to ovulation. It was found that ovulation occurred between 24 and 36 h after onset of oestrus in 54.6% of cycles (normal) and between 36 and 60 h after onset of oestrus (delayed) in the rest of the cycles (45.4%). Thus, delay in ovulation seems to be a major setback in the fertility of dairy cattle. Based on this ultrasonographic documentation, double inseminations at 24 h interval are being recommended in all the oestrus animals to ensure better conception rate.

2.3.3 Diagnosis of Pregnancy

Early pregnancy diagnosis is a key factor in the reproductive management of dairy herd. Timely diagnosis of the non-pregnant status of an animal is essential to rebreed at the subsequent oestrus and to maintain an optimum calving interval. A variety of methods were designed to identify the pregnant animal in the earlier stages.

• 1. Indirect Methods include hormonal, immunological, and chemical approaches.

• 2. Direct Methods include transrectal palpation and ultrasonography.

In human, hormonal estimation has been standardized for early diagnosis of pregnancy. In animals, especially in cattle, lots of pregnancy associated biomarkers are being identified but there lies an ambiguity in precise confirmation of pregnancy till date. Hence, direct methods are found to have advantage over indirect methods in the current scenario. Among the direct methods, the non-pregnant animals can be identified at least 15–30 days earlier by using ultrasonography when compared to standard transrectal method of examination.

A minimum of 60 days in the post-breeding period is required to diagnose pregnancy through rectal palpation. However, experienced and skilled veterinarians can confirm pregnancy at an early stage of 40–45 days post-breeding by the palpation of amniotic vesicle and double-slipping of chorioallantoic membrane. Several studies have suggested that examining pregnant cows early in gestation, especially by inexperienced technicians increases the risk of iatrogenic embryonic mortality. While rectal palpation is reliable and a widely accepted method for pregnancy diagnosis, it is difficult to assess embryonic viability and/or detect pregnancy loss with this technique.

Positive diagnosis of pregnancy is possible as early as 25 days post-insemination by the application of ultrasonography. Non-regressed CL (>15 mm diameter) by the period between 20 and 25 days post-insemination can be considered as the first indicator of pregnancy; however, it is not confirmatory due to various pathological conditions. As the conceptus develops, fluid accumulates within the allantoic and amniotic cavities during early gestation. Careful ultrasonographic examinations in the tip of the uterine horn, ipsilateral to the ovary having the CL, enable the examiner to identify this fluid accumulation as anechogenic zone along with the embryo (Fig. 2.8). Early pregnancy diagnosis using ultrasonography shows the uterine lumen containing a variable amount of. Identification of prominent CL and uterine fluid by 20–22 days and embryonic mass with heartbeat between 24 and 30 days post-breeding are considered as positive signs of pregnancy in cattle. These features make the ultrasonographic intervention a reliable method of early pregnancy diagnosis in cattle.

Fig. 2.8

Ultrasonographic imaging of uterine fluid and embryo by 24 days post-insemination

Scanty fluid accumulation could be observed in the uterine horn during oestrus also, due to physiological secretions and it should not be mis-diagnosed as pregnancy. How to differentiate it from early pregnancy? During oestrus, the mucus membrane folds are evident in the uterine endometrium of crossbred cattle, but the fluid-filled lumen of pregnant uterine horn possesses smooth lining without any ‘crenulation like’ pattern (Fig. 2.9).

Fig. 2.9

Uterine endometrial pattern during oestrum and early pregnancy. (a) Oestrum, (b) Early pregnancy

If an animal is presented for insemination with oestrus signs, which was inseminated in the previous cycle, it should be subjected for ultrasonographic investigation. If anechogenic fluid with smooth luminal wall could be observed in the tip of any uterine horn along with a prominent CL in the ipsilateral ovary, it should be suspected for early pregnancy and insemination should be avoided. Sometimes early pregnant animals evince oestrus signs due to presence of DF at that time. If such animals are inseminated, we will probably lose the pregnancy due to accidental induction of embryonic mortality. This might be one major reason for pregnancy loss in field conditions, where ultrasound examination is not practiced.

• The importance of ultrasonographic investigation of utero-ovarian structures before attempting for insemination in animals with oestrus signs is reiterated.

Mucometra and pyometra are generally defined as an accumulation of mucus and pus within the uterus, respectively. Compared to manual palpation, the differences between uterine enlargement due to pregnancy and these pathological conditions are easily recognizable on ultrasound examination. While fluid in the pregnant uterus appears anechoic, pyometra appears as distension of the uterine lumen with contents of mixed echogenicity. In case of mucometra, uterine fluid appears clear and anechoic like that of pregnancy, but there are no evidence of foetus, foetal membranes or placentomes on ultrasound examination (Fig. 2.10). Thus, ultrasound technology is the reliable method of diagnosing pregnancy and also to differentiate it from other pathological conditions.

Fig. 2.10

Ultrasonographic imaging of mucometra and pyometra (a) Pyometra, (b) Mucometra

2.3.4 Assessment of Gestational Age

Two methods can be used to determine the gestational age of the embryo or foetus. First method is based on direct measurements of the embryo or foetal dimensions, and the second method is based on the first appearance of certain embryonic or foetal structures.

Ultrasonographically, four different measurements can be made based on the view of the embryo/foetus. They are crown–rump length (CRL), trunk diameter, head diameter and head length.

• CRL is measured from the tail head to the crown of the skull.

• Trunk diameter is the widest part of the ribcage at the level of the umbilicus.

• Head diameter is the maximum diameter of the skull just caudal to the eyes.

• Head length is measured from the top of the cranium to the tip of the nose.

CRL is the earliest possible measurable parameter before the head and trunk portions can be distinguished. Until 50 days of gestation, a simple formula can be applied for assessing gestational age as follows:

$$ \mathrm{Length}\ \mathrm{of}\ \mathrm{the}\ \mathrm{Embryo}\ \mathrm{or}\ \mathrm{Foetus}\ \left(\mathrm{Mm}\right)+18\kern0.37em =\mathrm{Embryonic}\ \mathrm{or}\ \mathrm{Foetal}\ \mathrm{Age}\ \left(\mathrm{Days}\right) $$

For foetal ages greater than 50 days, ‘gestational chart’ should be used (Table 2.1). Some ultrasound units are equipped with inbuilt software to calculate the gestational age based on foetal biometry.

Table 2.1 Gestational chart for cattle

Curran et al. (1989) standardized the assessment of gestational age based on visual evaluation of first appearance of certain embryonic/foetal features, as mentioned in Table 2.2.

Table 2.2 Gestational age based on embryonic/foetal characters

2.3.5 Foetal Gender Determination

Ultrasonographic gender determination of the bovine foetus was first described by Curran et al. (1989). It was found that the relative location of the genital tubercle (GT), which will become the penis in the male and the clitoris in the female, could be used to determine foetal gender beginning at day 55 of gestation. By this stage, the GT has reached its final location immediately behind the umbilicus in the male or immediately under the tail in the female (Fig. 2.11). Until about 80 days of gestation, the GT usually appears as a highly echogenic lobed structure in both genders.

Fig. 2.11

Foetal gender determination based on position of genital tubercle—female foetus

2.3.6 Diagnosis of Abnormal Pregnancies

Ultrasound investigation addressed many gestational abnormalities like multiple pregnancies (Stroud 1994), early embryonic death (Fissore et al. 1986), and dead foetus (Fissore et al. 1986). It can also be used to monitor the conceptus that are poorly developed for their gestational age and pregnancies in which there is embryonic resorption or foetal abortion. There are, however, a number of ultrasonographic features of normal pregnancies that may mimic disease, and these must be recognized.

Foetal viability can be easily assessed with ultrasound. Foetal heartbeat can be observed clearly from the beginning of about 24 days of gestation. The foetal heartrate (FHR) should be at least 130 bpm in early gestation, increasing to about 190 bpm by 60 days. The amniotic and chorioallantoic fluids should be clear and anechoic until at least 70 days of gestation. After 70 days, the fluids of pregnancy may begin to appear cloudy in normal pregnancies. If the fluid is cloudy before 70 days and there is no detectable heartbeat, embryonic/foetal mortality can be suspected and it is advisable to recheck pregnancies. Separated chorioallantoic membranes may also be seen in cases of late embryonic or early foetal death, often appearing to be floating in the pregnancy fluids (Fig. 2.12).

Fig. 2.12

Empty chorioallantoic membrane detected by 80 days pregnancy

Dead foetuses often are very heterogeneous, with many areas of high echogenicity that do not correspond to normal bony anatomy. Foetal mummies are easily identified by echogenic bony remnants, nearly complete lack of intrauterine fluid, lack of foetal movement and heartbeat (Fig. 2.13).

Fig. 2.13

Mummified foetus

There is a lack of well-defined studies to determine the health status of a bovine foetus during the last trimester of pregnancy. The ultrasonographic examination of the late-term pregnancy in cattle consists of collecting pertinent information on the FHR, foetal activity, mean placentome size, thickness of the allanto-amniotic membrane, the uterine adnexa and dam health by transabdominal or transrectal ultrasonography (Buczinski et al. 2007). Transabdominal ultrasonography allows for diagnosis of abnormal placentomes and hydrallantois. Foetal inactivity or large hyperechoic particles imaged within the foetal annexes are associated with foetal distress or death, and should be reassessed to confirm compromised pregnancy. However, more extensive studies are needed to increase the level of understanding of bovine late pregnancy. Assessment of these parameters should be included in the bovine foetal biophysical profile. The ultimate goal of this knowledge is to decrease neonatal morbidity and mortality in low-and high-risk pregnancies (Buczinski 2009).

2.4 Role of Ultrasonography in Diagnosing Infertile Conditions

The use of ultrasound has proven to be a valuable tool for cattle breeders to assess valuable reproductive information beyond the scope of rectal palpation. Ultrasound investigation addressed many infertility conditions like cystic ovarian disease (Pierson and Ginther 1984), anoestrous (Satheshkumar 2021) and endometritis (Fissore et al. 1986).

2.4.1 Diagnosis of Cystic Degeneration

Cystic ovarian degeneration (follicular and luteal cyst) is an important ovarian dysfunction and a major cause of reproductive failure in dairy cattle with an incidence of 5.2 to 27.9% (Alexandra et al. 2008).

Ovarian follicular cyst (OFC) is a consequence of a mature follicle that fails to ovulate at the appointed time during the oestrous cycle. Earlier, OFCs were defined as fluid-filled structures of ≥25 mm in diameter persisting for 10 or more days on the ovarian surface (Roberts 1971). However, with the introduction of ultrasonographic imaging technology, it is determined that follicles of exotic and crossbred cows typically ovulate at 13–17 mm and 10–14 mm diameter, respectively (Ginther et al. 1996; Satheshkumar et al. 2012a, b). So follicles that persist at that diameter or greater may be considered to be cystic. Silvia et al. (2002) defined OFCs as fluid-filled follicle like structures, with a minimum diameter of 17 mm and persisting for more than 6 days in the absence of a CL and clearly interfering with normal ovarian cyclicity. OFCs have a peripheral wall thickness of <3 mm (Fig. 2.14) and are accompanied by a plasma progesterone (P₄) concentration of ≤1 ng/ml.

Fig. 2.14

Follicular cyst

Luteal cysts are also found to be fluid-filled structures but they have a peripheral wall thickness of ≥3 mm (Fig. 2.15). Further, the serum P₄ concentration is >1 ng/ml. Ovarian luteal cysts should not be confused with a physiological cystic corpora lutea containing a central fluid-filled cavity.

Fig. 2.15

Luteal cyst

By the routine rectal palpation, the examiner usually experiences difficulty in differentiating between follicular and luteal cysts. Evaluation of the cysts by ultrasonographic imaging can be used to diagnose between the types of cysts. The following ultrasonographic features of the cysts can be used to differentiate them with precision.

• The persistent anechoic structure of >17 mm diameter with <3 mm wall thickness is considered as follicular cyst.

• The persistent anechoic structure of >17 mm diameter with ≥3 mm wall thickness is considered as luteal cyst.

Web like intra-cystic fibrous strands could be observed in chronic cases of follicular and luteal cysts. This might be attributed to changes in coagulating proteins of the entrapped fluid as reported by Satheshkumar (2021).

In the recent years, an increase in the incidences of non-steroidogenic large persistent cysts is observed in crossbred cattle. This category of cysts is thin walled, fluid-filled structures uniquely characterized by their uncontrollable acute or chronic increase in diameter and co-existing along with the normal DF and CL (Fig. 2.16). No nymphomaniac or anoestrus symptoms could be observed in the affected animals, instead the cows are cyclic with normal follicular turnover but remains infertile (cyclic non-breeders or repeat breeders). They are extremely non-responsive to regular hormonal therapies and hence referred as ‘non-responsive follicular cysts (NRFC)’. Probably, their incidence came to limelight due to the regular application of ultrasonographic diagnostics. These non-responsive cysts are inert structures, and the only remedy is their mechanical evacuation. Ultrasound guided trans-vaginal or trans-gluteal route of follicular cyst evacuation was successfully reported in crossbred cows (Satheshkumar 2018a, b; Satheshkumar et al. 2018). Following cystic evacuation, animals are subjected for programmed breeding protocol, and conception rate of 66.7% was recorded in affected animals (unpublished data).

Fig. 2.16

Non-responsive follicular cyst along with the normal dominant follicle

Thus, ultrasonography proved to be a part of therapeutic approach alongside its inevitable role in diagnostics.

2.4.2 Anoestrum

Postpartum anoestrus (PPA) is an ovarian dysfunction which delays the resumption of cyclicity after parturition and is attributed to many factors ranging from negative energy balance to stress of any origin (Opsomer et al. 1996; Kumar et al. 2014). Ovaries of acyclic cattle were often referred to be inactive (Roberts 1971). However, intervention with ultrasonography revealed that acyclic animals also displayed variable degree of ovarian activity, characterized by follicular turnover but without expression of oestrus signs and ovulation (Satheshkumar 2021).

In acyclic animals, both the ovaries exhibit extensive follicular activity, and the DFs develop in a wave-like fashion similar to that of normally cycling animals. A maximum of two waves occur within a span of 25 days, with each wave extending for a period of 10–14 days. The follicular wave exhibits coordinated growth, static and regression phases as that of cycling cows. The DFs reach a diameter of >10 mm on par with the size of a normal ovulatory follicle (Fig. 2.17) and remain static for a prolonged period before entering the regression phase. The deficient metabolic and endocrine status of the follicular microenvironment would have affected the steroidogenic capacity of the follicular cells and the oestrogen production thereon. Development of oestrogenically inactive large follicles and lack of ovulation are found to be the major factors for the incidence of PPA in crossbred cows. Failure of the intra-follicular IGF/IGFBP system might prevent the follicular maturation and hinder the normal cascade of events towards ovulation leading to non-expression of oestrus signs and anovulation in acyclic animals (Satheshkumar et al. 2019a). Thus, uninterrupted follicular growth and atresia of follicular entities without culminating in ovulation are documented with the application of ultrasonography.

Fig. 2.17

Normal follicular turnover in both the ovaries of acyclic cows. (a) Right ovary with a dominant follicle, (b) Left ovary with a follicle

2.4.3 Endometritis

Endometritis is a common condition affecting dairy cattle, and it negatively affects reproductive performance. Clinical endometritis is characterized by the presence of purulent or muco-purulent uterine discharge (Sheldon et al. 2006), and about 15% of dairy cows have clinical signs of such uterine disease that persist beyond 3 weeks postpartum. Subclinical cases of endometritis are the inflammation of the uterine endometrium without any overt expression of clinical signs and characterized by clear oestrual discharge. Nearly 30–35% of dairy cows are affected by this subclinical infection between 4 and 9 weeks postpartum. (LeBlanc 2008) and thus fertility is negatively affected. Transrectal ultrasonography may be used to evaluate cows for signs of endometritis. On ultrasound investigation, accumulation of intrauterine fluid containing echogenic particles and thickening of the endometrium due to endometrial oedema and inflammation are considered to be the indicators of endometritis. However, ultrasonography alone does not always provide a definitive diagnosis of endometritis.

2.4.4 Diagnosing Congenital or Acquired Anomalies

Diagnosis of congenital anomalies in heifers is a determining factor to decide upon its breeding capability in the earlier ages. Even though anoestrus associated with utero-ovarian agenesis/aplasia could be detected by routine per-rectal examination, conditions like persistent hymen can be noticed only when the animal becomes cyclic. Massive accumulation of secretory fluid within the uterine lumen and vaginal canal is the alarming features in such anomalies (Fig. 2.18) which requires immediate intervention.

Fig. 2.18

Ultrasonographic imaging of uterus and cervix with accumulated mucus due to blockage of vaginal passage

Congenital or acquired peri-ovarian anomalies and their effect on the mammalian fertility is a subject of interest for many decades due to its scientific and clinical importance. It is proved beyond doubt that ultrasonographic investigation is the best option for diagnosing such anomalies of delicate peri-ovarian structures. Physical and physiological relationship between ovary, bursa and oviduct plays a major role in gamete capture, transport and fertilization. Compromises in ovario-bursal and oviductal structures due to inflammation and adhesions lead to reproductive failure ranging from temporary infertility to permanent sterility. Severe ovario-bursal adhesions affect the ovulation process by masking the ovarian surface. Similarly occlusion of fallopian tube due to inflammation or fibrosis leads to fertilization failure or tubal entrapment of fertilized embryos (Shivhare et al. 2012). Animals with bilateral ovario-burso-tubal adhesions are sterile and their diagnosis in the earlier stages is warranted (Satheshkumar et al. 2019b). Hyperechoic borders of the ovario-burso-tubal structures surrounded by anechoic fluid accumulation indicate the severity of inflammation (Figs. 2.19, 2.20, 2.21).

Fig. 2.19

‘Cystic ovarian bursa’ encapsulating the ovary

Fig. 2.20

‘Tubo ovarian cyst’ with fluid accumulation in fallopian tube

Fig. 2.21

‘Ovario-bursal’ adhesion

Ultrasonographic investigation provides the actual degree of affection and the fate of future reproductive capability of that particular animal.

2.5 Utero-Ovarian Haemodynamics and Doppler Imaging

The blood supply to any organ is closely related to the function of that organ and reproductive tract is no exception. Consequently, assessment of blood flow characteristics is an important evaluation parameter of reproductive function. Colour Doppler imaging ultrasonography is routinely used in human gynaecology and obstetrics as the diagnostic gold standard. In the recent years, the use of Doppler imaging is gaining momentum in veterinary practice also. Colour Doppler imaging provides important information about the physiological/pathological status of follicles and CL supporting clinical diagnosis and therapeutic decisions. Several studies have demonstrated the relationship of blood flow and utero-ovarian function throughout the oestrous cycle and pregnancy (Acosta et al. 2002, 2003; Miyamoto et al. 2006; Herzog et al. 2010; Satheshkumar et al. 2012b; Satheshkumar 2018b).

2.5.1 Basic Principles of Doppler Ultrasonography

Qualitative assessment of blood flow in capillary networks draining reproductive organs can be carried out non-invasively by colour Doppler imaging ultrasonography, and the quantitative parameters of blood flow can be assessed by pulsed/spectral Doppler ultrasonography (Ginther 2007a, b). Blood flow to the organ is displayed in colour superimposed on a two-dimensional, grey-scale ultrasonographic image (colour Doppler) or as a graph depicting pulsed Doppler-spectral analysis of blood velocity in a small area of a large blood vessel (spectral Doppler).

Transducer should be placed as close as possible to the ovary, and colour blood flow mapping of the ovarian structures is conducted in various transverse sections. In the ultrasound equipment, activation of the colour Doppler mode enables the examiner to identify the blood flow as coloured signals on the ovaries, tubular genitalia and the blood vessels in the adjacent region like middle uterine arteries, ovarian arteries, etc. The colour mode determined the direction of blood flow and the blood flow area within the structure. Basically, the Doppler signals are viewed in a range from red to blue colour. The variations in colour signals do not represent the arteries and veins, but it represents the direction of blood flow (Fig. 2.22).

• When the blood flow towards the probe, it appears as red to orange in colour.

• When the blood flows away from the probe, it appears as blue in colour.

Fig. 2.22

Colour Doppler signals according to the direction of blood flow

Since the uterine (mesometrial, endometrial and myometrial) and ovarian (mesovarium, luteal and follicular) arteries have a tortuous anatomy, blood flow usually appears as red and blue colour adjacent to each other.

The arteries and veins can be differentiated based on spectral Doppler study. Normally, the arteries will be evincing pulsations but the veins are devoid of pulsation.

Switching over to pulsed wave spectral Doppler mode, the Doppler gate is positioned over the blood vessel and a spectral mode waveform is taken for calculating an array of velocimetric indices recorded during the systolic and diastolic stages of the cardiac cycle. In the spectral mode, blood flow velocity variations are represented as a graphic wave form called spectrum (Fig. 2.23). By convection, waveforms above and under the baseline indicate the red blood cells moving toward and away from the transducer, respectively. The spectrum provides peak systolic (PSV), end diastolic (EDV), and time-average maximum (TAMV) velocities in m / sec or cm/sec (Ferreira et al. 2011). Doppler pulse duration (DPD) can be recorded by measuring the linear distance between two subsequent EDV points and is expressed in milli seconds—ms (Satheshkumar et al. 2013; Satheshkumar 2018b).

• PSV is the maximum point along the length of the spectrum.

• EDV is the ending point of the cardiac cycle.

• TAMV is the average of spectral maximum blood flow velocities.

• DPD is the duration of a cardiac cycle and is indirectly proportional to the pulse frequency.

Fig. 2.23

Spectral waveform and various velocimetric indices

Apart from these velocity and duration indices, Doppler device automatically calculates pulsatility index (PI) and resistance index (RI) based on inbuilt formulation. These indices have negative correlation with the vascular perfusion of the tissue downstream from the sample gate and indicate the resistance to the blood flow. A PI and RI value of <1 usually reflects the decreased resistance and free flow of the blood supply to any organ.

Intensity of the follicular and luteal blood flow is graded by visual evaluation of the proportion of the peri-follicular and CL area filled with colour Doppler signals and is subjectively expressed as low, medium, high and very high (Kaya et al. 2017).

The knowledge of ovarian vascular development is of fundamental importance to understand the processes that ensure the reproductive success. Continued re-modelling of the ovarian vascular network supports the cyclic development and regression of follicular and luteal structures throughout the reproductive life. The uterine branch of the ovarian artery is prominent and forms a utero-ovarian arterial anastomosis between the uterine and ovarian arterial systems. Severance of the utero-ovarian arterial anastomosis on the CL side during the oestrous cycle interferes with ovarian function more than severance of the ovarian artery (OA) indicating the importance of the anastomosis to CL blood flow. Further, the ovarian angiogenesis or neovascularisation is a complex process in which a delicate balance between promoters and inhibitors is maintained. Disturbance of this balance may result in a disrupted physiologic state or various pathologic conditions.

2.5.2 Follicular Vascularity

Follicles require coordinated vascular development and adaptations as their development advances towards ovulation (Martelli et al. 2017). Active angiogenesis or neovascularization of follicles provides important signalling for the development and steroidogenic functions (Robinson et al. 2009). Follicular angiogenesis and its related outcome on dominance, atresia and ovulation were recorded in exotic (B. taurus) cows and heifers (Acosta 2007; Siddiqui et al. 2009; de Tarso et al. 2017) and in crossbred cows (Satheshkumar et al. 2013; Satheshkumar 2018b).

2.6 Peri-Follicular Blood Flow and Its Effect on Ovulation

Blood supply to individual follicle is critical for allowing it to acquire ovulatory capacity (Acosta et al. 2003). Regulatory role of vasoactive substances released during luteinizing hormone (LH) stimulation had an important role in the ovulation process. Subsequent reports also related the importance of pre-ovulatory follicular vascularity to the conception rate and fertility (Siddiqui et al. 2009; Varughese et al. 2013).

2.6.1 Intensity of Blood Flow

The sequential follow up of peri-follicular Doppler signals indicating the intensity of blood flow revealed that the ovulatory follicle (OF) was well vascularized with consistent blood flow signals surrounding the base of the follicular antrum, while the adjacent subordinate or atretic follicles were devoid of detectable blood flow (Fig. 2.24a). The intensity of peri-follicular blood flow increased as the time of ovulation approached which was evident by enriched colour Doppler signals covering over 70% of the OF circumference (Fig. 2.24b). The increased intensity of peri-follicular Doppler signals indicated that there was an active neovascularization process and increasing arteriolar network encasing the OFs (Ginther 2007a, b). The supply of hormones, growth factors, nutrients and oxygen is ensured as a result of having an adequate blood supply to the follicles. Thus, it is confirmed that there is a strong association between the angiogenic/neovascularization process and the selection of the OF during the final phases of development in cattle (Varughese et al. 2017; Satheshkumar 2018b).

Fig. 2.24

(a) Pre-ovulatory follicle with peri-follicular blood flow signals at the base of the follicle in the initial stages (Note: subordinate follicles without detectable signals) and (b) Doppler signals covering over 70% of follicular circumference

2.6.2 Blood Flow Parameters in Relation to Ovulation

The blood flow not only determines the selection of the OF but it also have a definitive role on the ovulation. Blood flow velocity parameters and indices of the peri-follicular blood flow of the OF were studied from the day of induced oestrus (Day 0) until the time of ovulation. The follicular size and the circumferential blood flow area gradually increased until the time of ovulation (Palmer et al. 2006).

As mentioned earlier, ovulation is delayed in 45.4% of oestrous cycles in crossbred cows. Interpretation of sequential changes revealed that the mean DPD (874.3 ± 57.0 ms) and PI (0.62 ± 0.01) values were less on the onset of oestrus (Day 0) in the animals that ovulated normally (Fig. 2.25a) when compared to the animals which ovulated late (1140.6 ± 27.5 ms and 1.28 ± 0.15, respectively) (Fig. 2.25b). The decreased DPD and PI values in the former category are indicative of a relatively rapid pulse frequency and greater vasodilation and vascular perfusion (Anteby et al. 1996; Palmer et al. 2006; Siddiqui et al. 2009). Scanning electron microscopic observations of ovarian corrosion casts by Macchiarelli et al. (2006) confirmed the fact that capillaries of pre-ovulatory follicles are dilated to a greater extent than that of earlier developing follicles. Based on ROC analysis, it is determined that 929 ms is the optimal cut-off point for DPD and values lesser than this point indicates the normal ovulatory status and vice versa with higher values (Satheshkumar 2018b).

Fig. 2.25

Measurement of Doppler pulse duration (DPD): (a) Normal ovulatory follicle with rapid pulse frequency and (b) Follicle of delayed ovulation with slow pulse frequency

It is confirmed that the cascade of events induced by the LH surge in pre-ovulatory follicles was closely associated with a local increase in the peri-follicular blood flow, a functional adaptation that is important for the impending ovulation to occur.

• The lesser DPD (<929 ms) and PI (<1) values on the day of oestrus are positive indicators of normal ovulation process and any deviations in these blood flow parameters delayed the timing of ovulation.

This finding is a valuable diagnostic parameter and is an important consideration for future studies involving regulation of follicular vasodilation to hasten the ovulation process in cattle.

2.7 Luteal Vascularity

The CL is a site of intense angiogenesis and is the most vascularized structure in the body (Acosta et al. 2002). The luteal vascularity enables the luteal cells to obtain the nutrient substrates and hormone precursors that are indispensable to support P₄ biosynthesis and also to release the synthesized steroid into the general circulation (Miyazaki et al. 1998; Fraser and Wulff 2003). The nature of vascularity reflected the functional status and steroidogenic capacity of CL (Herzog et al. 2010; Kaya et al. 2017).

2.7.1 Luteal Vascularity During Normal Cycle

In a sequential study luteal haemodynamics throughout the oestrous cycle from the onset of oestrus (Day 0), blood flow could be observed as less intense sparse colour Doppler signals around the periphery of the luteal tissue from Day 3, which increased gradually and covered more than 70% of the luteal circumference during the mid-luteal phase (Day 9–10). The progressive increase in luteal blood flow could be attributed to the increasing endocrine activity of the structure. Herzog et al. (2010) and Kaya et al. (2017) recorded a significant correlation between the proportion of luteal blood flow and P₄ secretion. The levels of VEGF and FGF, the pro-angiogenic factors, which are involved in neovascularisation after ovulation are found to be elevated during luteinization and throughout the development of the CL during mid-luteal phase, confirming the role of luteal vascularity (Zalman et al. 2012). An intense increase in blood flow is appreciated between Day 15 and 17 of the oestrous cycle, which can be appreciated by the penetration of colour Doppler signals deep into the luteal parenchyma. This unique feature is correlated to the pulsatile release of prostaglandin (PG) from the uterus as indicated by drastic increase in the plasma PGFM concentration during the period (Miyamoto et al. 2006). After that period, the blood flow signals decrease and found to be restricted to the periphery as the CL, which started regressing towards the final stages of the cycle (Day 19–21). Thus, the increased vascularization of CL during Day 15–17 of the cycle was the first indication of initiation of luteolytic phenomenon (Fig. 2.26).

Fig. 2.26

Sequential changes in luteal blood flow during normal oestrous cycle (Day 3–4), Day 5–6, Day 9–10, Day15–17, Day19–21

Programmed induction of luteolysis is possible by administration of exogenous PG during the mid-luteal phase, but the early CL (till Day 4 after oestrus) is refractory to PG treatment (Beal et al. 1980). The non-responsiveness of early CL was suggested to be due to luteal insensitivity or insufficient numbers of PG receptors (Duchens et al. 1994). PG exerts its luteolytic effects by binding to plasma membrane receptors (Sakamoto et al. 1994) and apoptosis of luteal cells is caused by reduction in blood flow and hypoxia induced inhibition of steroidogenic enzymes. Although these events contribute to the later stages of luteolysis, little is known about the cascade of physiological events initiating the luteolytic process. Colour Doppler imaging has given a solution for this gap of information. Acosta et al. (2002) and Shrestha and Ginther (2011) observed an acute increase in luteal blood flow in response to a conventional dose of PG administered in both the early-luteal phase (Day 3) and the mid-luteal phase (Day10). However, Satheshkumar et al. (2014b) documented that the increase in vascularity in response to exogenous PG was restricted only to the periphery of the CL in the early-luteal phase group, while the deeply radiating property of the vascular perfusion was recorded in mid-luteal phase group. Investigations on luteal blood vessels by Shirasuna et al. (2008) revealed that microcapillary vessels were present in both the peripheral and central regions of early and mid-luteal phase CL, but large blood vessels (>20 μm) were abundant in the periphery of the mid CL when compared to the early CL. Miyamoto and Shirasuna (2009) suggested that PG stimulated endothelial Nitric Oxide Synthase—Nitric oxide (eNOS-NO) system caused the vasodilation of peripheral blood vessels of the mid CL. The eNOS-NO system would have dilated the microvascular bed within the mid CL, resulting in free flow of blood deep into the luteal parenchyma. This vascular perfusion would have enabled the luteolysin to reach the deeper luteal cells and initiated the luteolytic process in mature mid-CL, rather than the immature early CL. This salient difference in the vascularity between the two stages of development might be the determining factor that initiates the lysis in response to exogenous PG in crossbred cattle. In natural cycle, gradual increase in blood flow could be observed for an extended period of 2–3 days before spontaneous luteolysis due to the pulsatile release of PGF at sequential intervals (Ginther 2007a, b), but in PG treated cycles the vascular perfusion episode was acute.

2.7.2 Luteal Vascularity During Pregnancy

A functional CL is required for establishment of pregnancy because P₄ supports pre-implantation embryonic development enabling maternal recognition of pregnancy of farm animals (Grana-Baumgartner et al. 2020). Late embryonic mortality usually occurs between 18 and 42 days of gestation and contributed for 3.2–42.7% of pregnancy losses in cattle (Santos et al. 2004; Pohler et al. 2016). Hence, the endocrine competence of CL is very crucial during this period.

Visual analyses of luteal vascularity during the first trimester of pregnancy revealed that the intensity of blood flow was very high covering more than 90% of the luteal circumference and even reaching the deeper areas of luteal parenchyma between 25 and, 35 days of gestation and the high intensity is maintained with the advancement of pregnancy (Fig. 2.27). This high intensity of luteal blood flow in the early stage of pregnancy is suggestive of high degree angiogenesis and the increased blood supply from the ipsilateral uterine artery and ovarian artery (Pinaffi et al. 2018). The high intensity of luteal blood flow during the early stage of gestation might be a supportive mechanism for synthesizing and releasing optimum levels of P₄ in order to sustain the pregnancy. It could be inferred that active neovascularization, expanded luteal vascular bed and increased pulse frequency of luteal blood flow during the first trimester of pregnancy allowed adequate blood supply to the CL and thus supported the endocrine function and early embryonic/foetal survival.

Fig. 2.27

Intensity of luteal blood flow during various stages of early pregnancy, Day 25–35 of pregnancy, Day 45–60 of pregnancy, Day 60–90 of pregnancy

Blood flow has been suggested to be more appropriate than size for CL function evaluation (Herzog et al. 2010) because CL vascularization plays a key role in regulating luteal function (Miyamoto et al. 2006). Evaluations of CLBF could, consequently, be useful to detect pregnancy failures or to predict pregnancy rates after embryo transfer or timed-AI.

2.8 Utero-Ovarian Haemodynamics in Disturbed Fertility

Colour Doppler imaging facilities have provided new insights into causative factors of the common fertility disorders in cattle. An altered blood supply to the uterus and ovaries could disturb the normal cycle and conception in cattle.

2.8.1 Blood Flow Parameters in Relation to Anoestrum

Blood flow in OA is related to the vascularity of intra-ovarian arteries and the ovarian follicles. The PI, RI and DPD indices of the OA ipsilateral to the ovary having the largest follicle were higher in anoestrus than in oestrus group of animals (Satheshkumar et al. 2017). Thus, increased flow resistance (indicated by high PI and RI) and slow pulsation (indicated by high DPD) of the blood flow in OA of anoestrus animals are the major factors affecting the vascular perfusion of follicles. Thus, altered blood flow parameters of OA are proved to be a predisposing factor for deficient follicular development and maturation in anoestrus cattle.

2.8.2 Blood Flow Parameters in Relation to Cystic Degeneration

Díaz et al. (2019) recorded marked changes in blood irrigation area of walls of persistent follicles simulating the early stages of development of follicular cyst. They found that the Doppler signals of blood flow were less in walls of persistent follicles than in normal DFs (Fig. 2.28a). As described earlier, there will be variation in the wall thickness between the follicular and luteal cyst. The Doppler technology shows the blood flow in the wall, and the area of the blood flow therefore gives a clearer view of the thickness of the wall (Fig. 2.28b).

Fig. 2.28

Vascular pattern in follicular and luteal cysts, (a) Follicular cyst, (b) Luteal cyst

2.9 Conclusion

Growth and regression of ovarian structures in cattle can be monitored with ultrasound on a sequential basis and in a non-invasive manner. Ultrasound is more accurate than rectal examination in determining the dimensions of ovarian structures. Thus, the application of real-time ultrasonography for monitoring ovarian function in cattle has advanced the understanding of follicular and luteal dynamics and its regulation. Colour Doppler sonographic studies showed that it is a useful technique for the investigation of the utero-ovarian blood flow and provide new information about physiological changes during various phases of cycle. Based on the basic studies, it could be concluded that the control of angiogenic development in the ovary could be a useful tool to improve animal reproductive performances. In addition to manual per-rectal examination, ultrasonography should be included in the infertility investigation to examine and evaluate the reproductive status of cattle. This enables to assess the architecture of the ovaries, uterus, reproductive vasculature and surrounding structures for accurate diagnosis of the aberrations. Early diagnosis with appropriate therapeutic intervention will aid in improving the bovine fertility and production thereof.