Reproductive Application of Ultrasound in the Female Buffalo (Bubalus bubalis)

Reproductive ultrasound (RUS) has been successfully used in animal reproduction for the past three decades [1-3]. This tool emerged as an accurate, non-invasive and reliable technique for the early diagnosis of pregnancy in buffalo [4-6] and to elucidate follicular patterns of development [7-9]. RUS has gained popularity in buffalo reproduction during recent years as it has many research and clinical applications such as evaluation of ovarian function and CL development [10-12], fetal development and fetal sexing [13,14], uterine involution [15,16], evaluation of genital pathologies [17], and embryonic mortality during early pregnancy [18,19]. Insights gained via RUS on the reproductive events in buffalo has stimulated its use for application in more advanced reproductive techniques like monitoring of superovulation [20], trans-vaginal ultrasound guided ovum pick from live donors for in vitro fertilization [21-24], and fertility monitoring during artificial insemination and estrus synchronization programs [25-27]. In this chapter the basic concepts of reproductive ultrasonography, the common indications for the use of RUS and the diagnostic features of RUS are documented and the sonograms are presented for understanding.

1. Basic Concepts

Ultrasound covers the spectrum of sound frequencies exceeding 20,000 cycles/sec, which is the maximum frequency perceived by the human ear. The sound waves go to the tissues and are reflected back to the transducer (crystals). The ratio of reflected waves or echo is received and converted into electrical impulses, processed and displayed through an oscilloscope and cathode ray tube according to the type of ultrasound machine used [28-31].

The acoustic image or aligned echo depends on the property of reflection of the waves into the tissues together with the position of the ultrasonic beam. The ultrasonic beam causes the molecules in the tissues to vibrate; ultrasounds must overcome a specific resistance called acoustic impedance. The acoustic impedance may be estimated by multiplying the density of tissue by the propagation speed of sound, the average speed of the soft tissues is 1.540 m second. The boundary between two tissues of different impedance is called acoustic interface. The ability in the propagation of the sound depends on the distance between molecules, if the molecules are so close from one to another or if there are a greater number of molecules per unit volume, then the sound is transmitted faster. Each representation of the echoes that returns, is recorded on a video monitor, so obtained images could be then interpreted by the clinician [28,29,32,33].

1.1. Types of Ultrasound

-        Real time B-mode:

A two-dimensional diagnostic ultrasound presentation of echo-producing interfaces; the intensity of the echo is represented by modulation of the brightness (B) of the spot, and the position of the echo is determined from the angular position of the transducer and the transit time of the acoustical pulse and its echo. B-mode ultrasound provides lots of information in a short period of time, allowing a dynamic anatomic diagnosis. A two-dimensional image in a series of images in action that is generated to be produced quickly, gives an idea of movement structure analysis in real time. The signals are repeatedly transmitted, received and processed so continually has an updated image of the organ. This is the method most widely used for the examination of the reproductive tract of cattle and other large animals including the water buffalo [28,29,34,35].

-        A-Mode:

The so called amplitude mode produces a one dimensional display of echo amplitudes for various depths, depicted as a line graph where the axes are amplitude and depth. The principal use of the A-mode is to evaluate the fat and lean portions of meat animals. Although the A-mode has also been used for pregnancy diagnoses, the real time B-mode is the most popular for this purpose [28-30].

-        M-Mode:

The motion mode is an adaptation of the B-mode and is used to evaluate moving structures such as the heart. The change in reflector depth at different times is displayed as a simple line graph where the axes are depth and time [28-30].

-        Doppler:

Doppler systems use the motion of blood toward, away or at an angle to the transducer to construct dazzling multicolor images of flow patterns (red= toward the transducer; blue= away from the transducer): Doppler is useful to evaluate blood flow of the fetal heart, corpus luteum formation, monitoring ovulation among other clinical features [28,29,36-38].

1.2. Types of Transducers

Transducer refers to any device that converts energy from one form to another. Ultrasonic transducers convert electric energy into mechanical energy for production of ultrasonic waves and also convert the acoustic energy of echoes into electric energy. There are three types of arrays [28-30].

-        Linear array:

Linear arrays refer to the side by side arrangement of the rectangular electric crystals along the length of the transducer. The examining field and the two-dimensional image are observed on the screen in the shape of a rectangle. The rectangular field of a transrectal linear –array scan is oriented longitudinally with respect to the animal. Most of the ultrasound scanners used for rectal examination of the reproductive tract of large animals uses sequential linear array systems (Fig. 1A).

-        Sector (sectorial) array:

Refers to the pie-shape examining field and image. Sector transducers are useful to project beams through narrow openings, such as the space between ribs. The beam enters the tissues through a small window and then spreads out.

-        Convex:

 Convex are curved arrays that have been introduced to produce a sector like- field with resolution similar to that of the linear array. Convex transducers are very useful for transabdominal imaging in small animals or small ruminants, and for trasnsvaginal aspiration of oocytes in large animals (Fig. 1B).

Figure 1. Representation of the ultrasound beam. Panel A. Linear array, and Panel 1B. Convex array.

Improvement in manufacturing has allowed development of transducer crystals with a wide frequency spectrum, a broadband probe might provide the option of selecting frequencies from 2.0-10 MHz. Typically, sector and convex transducers require frequencies from 2.0-6.0 MHz, while linear probes require frequencies between 5.0-10 MHz.

2. Ultrasonographic Examination of the Non-Pregnant Reproductive Tract

2.1. Reproductive Anatomy

Water buffalo are considered seasonal polyestrous, mono-ovulatory, short-day breeders species. They exhibit a fairly seasonal variation in display of estrus, conception and calving rates. Although their anatomy and reproductive physiology resembles that of cattle, some differences are important to state for better understanding.

2.1.1. Uterus

The tubular genitalia of the buffalo are generally more muscular and firmer, and the uterine horns are more coiled than those of the cow. The body of the uterus is much shorter (1–2 cm) than that of the cow (2–4 cm). The cervix of the water buffalo is smaller than that of the cow (length 3–10 cm, diameter 1.5–6.0 cm) and its canal is more tortuous. The average number of cervical folds in water buffalo is three (Fig. 2) [39]. In addition, the broad ligament seems to be shorter and tighter compared to those in cows, which makes it sometimes difficult to fully retract and expose the uterine horns during routine rectal examinations of the non-pregnant buffalo female.

Figure 2. Longitudinal ultrasonographic image of the cervix of a female buffalo. Arrows point at the cervical rings.

2.1.2. Ovarian Anatomy

The buffalo gonad is of a smaller size and lighter weight compared with that of domestic cattle (2.5 cm vs. 3.7 cm of length; 3.9 g vs. 8.5 g of weight respectively) [40]. The morphological appearance of the CL has also been described [10], it has been observed that the CL is deeply embedded in ovarian stroma and it is smaller than the CL in cattle [41,42].

Diagnostic ultrasound allows the evaluation of morphological changes of the uterus during the estrous cycle [11], postpartum uterine involution [11,15,16,43,44], evaluation of ovarian biometry [11] as well as pathological conditions of the non-pregnant female [17]. It is important to emphasize, that in order to properly perform ultrasonographic examinations, the clinician has to have good clinical skills to perform rectal examinations to be able to correctly identify reproductive structures. Initially, a routine rectal examination should be performed in order to identify all the reproductive organs. The examiner must then completely evacuate the rectum and introduce the lubricated transducer through the rectum. The orientation of the transducer should be longitudinal, in the direction of the uterus. The transducer should be slowly moved toward the cranial vagina, then to the cervix, uterine horns, and finally, the examiner will direct the transducer laterally to reach the ovaries.

For the examination of the reproductive tract, the transducer should be placed dorsal to the structures (Fig. 3A). The cervix, uterine body and uterine horns can be observed on the monitor in an either longitudinal or transverse cross-section depending on the position of the transducer (Fig. 3A, Fig.3B, and Fig. 4). During the examination of the uterus, it is important to minimize the distance between the transducer and the examined structures, this requires applying gentle pressure with the transducer on the uterus to obtain the best possible image and to minimize trauma to the rectal mucosa.

Figure 3. Longitudinal view of the uterus of a non-pregnant buffalo, the small arrow shows the dorsal curvature of the uterus. 3B. Cross-sectional image of a non-pregnant uterus, the arrows are showing sagittal sections of the uterine horn.

Figure 4. Cross-sectional image of a uterine horn of a female buffalo during pro-estrus, it is possible to visualize the thickness of the endometrium and the lumen of the uterus showing some non-echogenic fluid. An ovarian follicle <8 mm diameter is also shown (Linear transducer of 6.5 MHz).

2.2. Evaluation of the Uterus

In order to adequately characterize the images obtained, it has been suggested to divide the uterine horn in four to five segments in order to be able to describe the location of an embryo or of a specific change that may suggest disease within a specific area when a longitudinal section of the uterus is observed (Fig. 3A). If the transducer is placed away from the longitudinal axis, then, the image obtained will show different cross sectional sections of the uterine horn (Fig. 3B).

Physiological changes are evaluated based on parameters such as echogenicity, vascularity and edema of the uterine wall as well as the accumulation of fluid into the lumen of the uterine horn. The echogenicity of the uterine wall increases during estrus due to the increased uterine tone. Vascularity and edema are characterized as non-echogenic areas within the uterine wall, and they denote the day of the estrous cycle dominated by estrogens. Occasionally, physiologic accumulation of fluid into the uterine lumen could be observed during the estrus in female buffaloes (Fig. 4) [11] as also observed in cattle [45,46]. Pathological conditions of the uterus will be covered later in this manuscript.

2.3. Evaluation of the Ovaries

For a proper examination of the ovary by ultrasound, it is important to emphasize that the transducer must be located in close contact with the ovary to avoid artifacts due to the small area of contact, which leaves most of the transducer free or in contact with other tissues. In order to obtain good quality images it is necessary for the clinician to manage the manipulation of the ovary to adequately differentiate the structures present at a certain time.

The image of a follicle observed in the screen is characterized by its round shape, commonly smaller than 8 mm, containing a non-echogenic fluid surrounded by a thin echogenic wall (Fig. 5A). On the other hand, the corpus luteum (CL) looks typically as an echogenic structure (the CL appears as a gray structure on the image) which range in size from 13-16 mm in diameter in its mature state depending on the day of the estrous cycle (Fig. 5B). As mentioned before, CL in buffalo are smaller compared to those of cattle [42], and they do not tend to protrude from the ovarian parenchyma which makes it difficult to positively identify them during the rectal exam. Size and presence of a CL are important to assess cyclicity of the animal, and thus, to initiate estrus synchronization protocols, embryo transfer treatments, transfer of embryos to recipients, or to diagnose anovulatory conditions, and true anestrus.

Figure 5. Ultrasonogram of two buffalo ovaries. A. Image of an 8 mm dominant follicle (DF), also shown a pool of growing follicles <3 mm diameter (White arrows), note the non-echogenic fluid-filled follicles. Figure 5B. Arrows show a corpus luteum (CL), note the echogenic texture of the ovary (Linear transducer of 7.5 MHz).

Although ultrasound examination is more sensitive than rectal examination to detect both CLs and follicles, it is important to keep in mind that a single ultrasonographic evaluation of the ovary does not provide a definitive diagnosis about which structure (follicular or luteal) is dominating at the time of the examination, and obviously, it is important to analyze in detail both ovaries and the individual history of the animal. Table 1 resumes some relevant information with regard to folliculogenesis in female water buffaloes.

2.4. Estrous Cycle and Follicular Dynamics

Water buffaloes are seasonally polyestrous with an average cycle length of 21 days (range 17–24 days), and an average duration of estrus of 18 h (range 5–36 h) [42]. Compared to cattle, estrus behavior in the water buffalo is more subtle, and homosexual mounting behavior is not common. Secondary signs such as swollen vulva, reddening of the vulvar mucosa, and frequent urination are not reliable indicators of estrus [47]. Typically, ovulation occurs 30 h after the onset of estrus (range 18–45 h). The interval from the end of estrus to ovulation has been estimated to range from 10 to 24 h [48-50]. In Murrah buffalo it was estimated that ovulation occurred at 42.2 ± 2.8 h with a range of 28 to 60 h after onset of spontaneous estrus and the diameter of an ovulatory follicle is about 15.5 ± 1.6 mm [51]. In addition, follicular deviation or selection of the dominant follicle (time at which a deviation in growth rate between the dominant and largest subordinate follicle takes place) occurred 2.6 ± 0.2 d after ovulation, when the dominant and the largest subordinate follicle reached 7.2 ± 0.3 and 6.4 ± 0.3 mm [52]. Although double ovulations are rare [42,47], a double consecutive LH peak, usually accompanied by a double ovulation was reported to occur [53]. In the event of a double ovulation followed by pregnancy, the fertilized oocyte apparently derives from the last ovulation [54]. The diameter of the mature CL ranges from 10 to 15 mm and the ovulation papilla, or crown of the CL does not protrude much beyond the surface of the ovary, making it more difficult to identify by rectal palpation. As observed in cattle, follicular growth occurred in waves in female buffaloes [51]. Two-wave cycles were most common (63.3%) followed by three-wave cycles (33.3%) and a single wave cycle (3.3%). The number of waves influenced the length of the luteal phase and the estrous cycle [51]. Some reports have documented the one-wave pattern of follicular development and suggested that such a pattern may be common in B. bubalis [55,56].

The ultrasonographic study of follicular dynamics has allowed the identification of patterns of follicular growth and development throughout the estrous cycle in water buffalo, which has been useful to identify one to three waves of follicular development. At the end of the estrous cycle the last wave will result in the ovulation of the last dominant follicle. Each follicular wave lasts around 11-12 days, a period in which follicles present a recruiting stage, followed by a selection of a follicle that becomes dominant and which causes atresia (regression) of the other follicles present in the cohort at that time. Animals with three follicular waves usually have cycles longer than those with two waves (Table 1) [56].

Knowledge of follicular patterns and folliculogenesis are necessary to successfully implement both estrus synchronization and superovulation protocols, as well to diagnose anovulatory conditions and anestrus in female buffaloes.

2.5. Diagnosis of Ovarian Disorders

Among other RUS applications, the diagnosis of anovulatory conditions has been reported in female water buffaloes [57]. Ovarian cysts are probably not as commonly seen in buffaloes as in dairy cattle, however, they can still be observed [57]. Different studies revealed a prevalence of cystic ovaries range 0.4 - 2.8 % [58,59]. Ovarian cysts are anovulatory, simple or multiple structures larger than 25 mm in diameter (Fig. 6) in the absence of a CL that alters ovarian cyclicity and therefore, bubaline fertility. In addition RUS is useful to assess ovulatory failure or determine whether the ovarian follicular cyst is either follicular or luteal for therapeutic purposes. Chronic cases may lead to uterine accumulation of fluids in the uterus (mucometra) (Fig. 6).

Figure 6. Ovarian Follicular Cyst (FC). UB: Urinary bladder. UH: Uterine horn showing accumulation of fluid in the uterine lumen (mucometra).

Other anovulatory conditions can also be evaluated by RUS. Follicular growth during anestrus can also be determined. It is necessary though, to implement an adequate follow-up to monitor persistency of anovulatory structures within time. Additionally, the occurrence of unilateral or bilateral hemorrhagic cysts (Fig. 7) has been reported previously [60].

Figure 7. Ultrasonogram of an ovarian follicular cyst of 35 mm diameter. A. The cavity is fully filled with fluid. A partially luteinized follicular wall (3 mm) can also be observed (Linear transducer of 5.0 MHz).

Parovarian cysts (POC) are remnants of the mesonephric ducts that are occasionally found around the ovary and oviducts, attached to the broad ligaments of both cows and buffaloes [61]. POC could be an incidental finding at the time of slaughter. These cysts may vary in size from 1 to 5 cm in diameter and are usually round or oval in shape with a central fluid–filled non-echogenic cavity. In addition, POC can sometimes be misdiagnosed as follicular cysts during a rectal palpation examination due to their close proximity to the ovary (Fig. 8). The prevalence of this condition has been reported to vary from 0.4 - 13.0% [62].

Figure 8. Ultrasonographic image of a large 30x35x30 mm Paraovarian cyst (POC). On the image, the ovary can also be observed on the right (OV) as well as a 5 mm follicle (F) (the dotted line corresponds to 10 mm in length).

The diagnostic approaches for ovarian dysfunction and ovarian pathologies include transrectal palpation and transrectal ultrasonography [57]. Due to the small size of the ovaries and therefore the ovarian structures, RUS have become a suitable tool to improve the efficiency of the diagnosis of ovarian conditions affecting female buffaloes. In addition, accurate estimation of time at ovulation by ultrasound has resulted in a higher pregnancy rate following estrus synchronization in buffalo.

2.6. Diagnosis of Uterine Pathologies

Ultrasound examination of the uterus is one of the most rapid, precise, and least invasive methods to evaluate postpartum uterine health [1,46,63]. Criteria for evaluation of the inflammatory conditions of the uterus may include: 1) thickness of the uterine wall, 2) detection of fluid in the uterine lumen, 3) changes in the vaginal secretions evaluated by vaginoscopy. The last criterion has been accepted as the most reliable test in dairy cows; however, this is not performed routinely in beef cattle or buffaloes.

Normal involution of the postpartum uterus can also be evaluated by both rectal examination and ultrasound (Fig. 9A). Sonographic evaluations revealed that the uterine involution is completed by Day 22-31 [15,16]. The main pathological conditions of the uterus that can be diagnosed using ultrasound are inflammatory disorders [17]. These include puerperal and clinical metritis, clinical endometritis, pyometra, and mucometra [17], as also observed in cattle [1, 63-65]. The definitions of these postpartum uterine conditions in buffalo [66] have been addressed and extrapolated from cows [67], and are useful to orient both the clinical diagnoses and the therapy.

Figure 9. Longitudinal section of the normal uterine involution of a buffalo cow at Day 15 postpartum, a slight edema represented by non-echogenic small areas within the uterine horn can be seen (arrows). Figure 9B. Image of the 5 day postpartum uterus of a buffalo with retained fetal membranes suggesting puerperal metritis. A uterine caruncle (right) surrounded by hyperechoic fluid into the uterine lumen is indicative of puerperal metritis.

The diagnosis of acute puerperal metritis is generally reserved for the first 10 to 15 days of lactation, although it may still occur up to 21 days postpartum [67]. Ultrasound confirmation is not necessary in cases of acute puerperal metritis if the female has already shown clinical signs (fetid watery red-brown uterine discharge, with signs of systemic illness, endotoxemia and fever). Ultrasound examination of clinical metritis usually shows a thick uterine wall, accumulation of fluid into the lumen with varying degrees of echogenicity and typically contains hyper-echogenic debris (Fig. 9B). Buffalo cows are not systemically ill but have an abnormally enlarged uterus and a purulent uterine discharge detectable in the vagina within 21 days postpartum.

Endometritis is a relatively frequent pathology in the postpartum buffalo cow with a reported prevalence of 8.9 % [58]. Endometritis reduces the reproductive performance of cattle and buffaloes; therefore, improving early diagnosis is essential to reduce economic losses. Diagnosis of endometritis by rectal palpation and eventually by the observation of a vaginal discharge, if present, is the most consistent clinical routine. Vaginal speculum examination improves the accuracy to detect endometritis. Nevertheless, the diagnosis of clinical and subclinical endometritis in buffalo is undertaken using a variety of techniques including rectal examination [62], vaginoscopy [68], endometrial cytology [69], hysteroscopy and ultrasonography [64,67,70,71].

Clinical endometritis is characterized by the presence of purulent uterine discharge detectable in the vagina 21 days or more after parturition. In the absence of purulent discharge, a female with accumulation of abnormal contents in the uterus can also be diagnosed as having a clinical endometritis. Pyometra is defined as the accumulation of purulent material within the uterine lumen in the presence of a persistent CL and a closed cervix [66]. Ultrasound examination of this condition shows a variable accumulation of purulent liquid that is non-uniform in echogenicity; with hyper-echogenic particles present [72].

RUS is used to evaluate the uterus; the degree of echogenicity of the intrauterine fluid suggests the presence of contaminated material. The uterine fluid can vary from non-echogenic (Fig. 10A) to echogenic depending on the different degrees of mucus or the presence of purulent material. As a general principle, as the level of echogenicity of the uterine fluid increases, the severity of the endometritis increases too (pyometra) (Fig. 10B). Also, when the uterine wall is compromised (metritis) it becomes more echogenic. Improved diagnosis of clinical and subclinical endometritis should be performed using endometrial cytology or endometrial biopsy [69].

Figure 10. Transverse section of the uterus in a non-pregnant buffalo with a non- echogenic fluid accumulation (mucometra). Figure 10B. The uterine fluid has a hyper-echogenic appearance (pyometra). Also, the endometrium shows increased echogenicity and thickness.

3. Embryo Transfer and Multiple Ovulation Programs

Embryo transfer techniques in water buffalo were derived from those in cattle [73]. During the early 1980s different treatments and gonadotrophin sources were tested for their ability to induce superovulation in buffaloes. Regardless of the source of gonadotropin and its dosage, the success rate has been much lower in buffalo than in cattle. The low success has been attributed to the buffalo’s inherent lower fertility, and poor superovulatory response to gonadotropins [74].

The objectives of superovulation include inducing a high number of ovulations and subsequent high fertilization rate of oocytes derived from these ovulations, while at the same time ensuring a normal physiological environment in the reproductive tract for embryo development. A major factor limiting extensive use of embryo transfer in buffalo is the unpredictability of the superovulatory response which has led to low embryo recovery rates. In a comparative study in which two commercial sources of Follicle Stimulating Hormone (FSH) were administered at different dose levels, the number of total recovered transferable embryos varied between 0.15 and 2.27 per buffalo cow [75], which is much lower than the reported average number of embryos recovered in cattle [76,77].

Even though the in vivo production of buffalo embryos in Multiple Ovulation Embryo Transfer programs (MOET) remains low, RUS has been successfully used to study the response to the superovulatory treatments [20,44], estimated ovulation and embryo recovery rates, and to determine folliculogenesis problems following gonadotrophin stimulations (Table 2). Significant advances were made after the introduction of ultrasound examination to assess folliculogenesis [8,78]. These studies helped to better understand the dynamics of growth, differentiation and maturation of ovarian follicles during the estrous cycle, with the aim of improving superovulation protocols, and therefore increasing embryo transfer application in buffalo [79]. The presence of a dominant follicle at initiation of superovulation lowers the subsequent superovulatory response and the number of recovered embryos [20]. Thus, ultrasonographic evaluation of ovarian structures prior to initiation of superovulation can be a useful approach. Table 2 presents the results of a study conducted to assess the superovulatory responses of female buffaloes [80].

Through endocrine and ultrasonographic studies it has been determined that buffaloes are capable of responding to the stimulation of the ovaries with gonadotropins. However, either they fail to ovulate or there is failure of transport of the fertilized oocytes to the uterus. Fig. 11 shows the response of a female buffalo during a superovulatory protocol with FSH. A comparison of Day 2 vs Day 5 of treatment in the same animal is shown. Panel 11A shows a CL in the right ovary and at least 10 follicles < 5 mm between the two ovaries. The effect of FSH on follicular development and growth can be observed in panel 11B, the CL is no longer present because of the use of prostaglandin F2 alpha on Day 3 and 4 of the protocol. It is also possible to observe a group of larger follicles > 8 mm that have already acquired ovulatory capacity, and therefore are eligible to be ovulated.

Figure 11. Ultrasonographic images of the ovaries of a superstimulated water buffalo 11A. Image of the two ovaries of a female buffalo on Day two of the FSH superovulatory treatment; a CL (a) and a group of follicles (b) < 5 mm diameter are shown. 11B. A scan of the same animal was performed at Day 5 after the FSH treatment started, a group of follicles between 8-10 mm were observed (b). Note the increased size of the ovaries.

It is essential to evaluate the superovulatory response on the day of embryo collection (Day 5.5 after artificial insemination) by ultrasonography. A fairly good response is shown in Fig. 12A. There is a moderate increase in the size of both ovaries coupled with the presence of at least twelve CLs between the right and the left ovary. Unfortunately, this is not the most common pattern in female buffaloes; thus without inducing ovulation as a part of the superovulatory treatment, it is common to find a single CL with some large retained follicles > 10 mm diameter (Fig. 12B). In this case, the superovulatory response is considered poor, and therefore, the embryo collection rate will be very low [81].

Figure 12. A. Ultrasound Image of a buffalo ovary with very good superovulatory response to gonadotropins, a total of eight CLs (a) are observed on the right ovary, while there are four CLs on the left ovary. 12B. Ultrasound image of a buffalo ovary with poor superovulatory response to gonadotropins, the right ovary has two retained follicles (b), while the left ovary has a single CL (a).

Although female buffaloes may respond to superovulatory treatments, the embryo recovery rate still remains low, therefore, different alternatives such as ultrasound guided follicular aspiration and in vitro fertilization have been studied.

4. Applications of RUS for in vitro Embryo Production in Buffalo Species

As a consequence of the low efficiency of superovulation and embryo transfer programs in buffaloes, there has been an increasing interest in in vitro embryo production (IVEP) technologies in recent years. The common method of IVEP involves oocyte culture, maturation, and in vitro fertilization (IVF), with frozen thawed semen and then incubation of the resulting cleaved embryos in culture until they develop into morulae or blastocysts which can be transferred non-surgically to recipients or cryopreserved for future use [82,83]. Nevertheless, the technique produces fewer viable embryos in buffalo compared to cattle [82,84] (Table 3).

4.1. Ultrasound Guided Follicular Aspiration

The combination of oocyte recovery from follicles using trasnsvaginal ultrasound guided follicular aspiration (OPU) and IVF techniques have become better alternatives for overcoming difficulties in buffalo embryo production [82,84]. A successful application of these combined OPU/IVF technologies in buffaloes has been reported. One study reported that 18.3% of oocytes aspirated eventually developed into transferable embryos; following non-surgical transfer of these embryos (n=20) two established pregnancies, were spontaneously terminated between 3-5 months of gestation [85]. Another study reported an average of 8 to 10 oocytes collected per OPU with an average production of two transferable embryos, but this study did not report pregnancy outcome [21].

To perform OPU, special equipment is necessary (Fig. 13) to allow the puncture of the ovary and at the same time the aspiration of the follicular contents. This technique includes the use of a trasnsvaginal convex probe (preferably 5-7.5 MHz) which has a guide to hold an 18 g needle attached to a vacuum pump system. Usually the system requires a negative pressure of 60-70 mm Hg to aspirate the oocytes without losing the granulosa cells, this is very important to achieve adequate cleavage rates. Moreover, the rectum must be evacuated previous to performing epidural anesthesia.

Figure 13. A. Vacuum pump, B. A 5.0 MHz convex transducer provided with an 18 g needle. C. Image of buffalo ovarian follicles (a) Follicles > 8 mm diameter, (b) a 3 mm diameter follicle. D. Image of ovarian structures, (a) corpus luteum, (b) a 3 mm diameter follicle, and (d) a cross-sectional view of the uterine horn.

Even though IVF has been shown to be a feasible technology for obtaining and multiplying superior genetic stocks, there are still some constraints, not only with regard to the relatively low numbers of embryos produced by OPU/ buffalo cows, but also constraints associated with the availability of recipients and the regulatory limitations on international trading of fresh IVF produced embryos [86].

5. Ultrasonographic Examination of the Pregnant Reproductive Tract of the Female Buffalo

RUS has been used widely for pregnancy diagnosis and fetal gender determination in different species [87,88]. Through the use of RUS fetal and placental development and viability can be monitored and embryonic or fetal mortality can be diagnosed and confirmed early. Traditionally, pregnancy diagnosis in buffaloes has been performed by rectal examination of the uterus similar to cattle; results are variable depending on the skill of the clinician.

5.1. Early Pregnancy Diagnosis

In general, as gestation progresses, the diagnosis by rectal examination becomes easier; however, to increase reproductive efficiency at the herd level, it is important to accurately identify non-pregnant animals, either repeat breeders or those who have experienced embryonic death. Early pregnancy diagnosis by RUS can efficiently detect problem animals at an early time to facilitate decision making in a timely manner in order to minimize economic losses generated by low pregnancy rates, increased days open and low calving rates. RUS is a non-invasive method that allows pregnancy detection in buffaloes at around Day 25-33 of gestation, [89] unlike transrectal palpation which allows early diagnosis between 35-60 days depending on the skills of the clinician [4,90]. Nevertheless, the results depend on the type of equipment used, quality of the image and resolution, type of transducer, and the experience and interpretation of the operator [91]. The results can also be influenced by the age of the animal and the calving number, making early diagnosis easier in heifers than in pluriparous animals.

Images of early pregnancy diagnosis by RUS, allows the visualization of non- echogenic fluid in the lumen of the uterine horn. The pregnant horn must be symmetrical and spherical, which corresponds to the allantoic cavity fluid (Fig. 14). The presence of an embryo inside the cavity confirms the diagnosis, but their viability can only be confirmed through visualization of the heartbeat (Fig. 15).

Figure 14. Ultrasonographic image of a 28 day old pregnancy in a water buffalo female. Corpus Luteum (CL), Urinary bladder (UB), and the spherical and symmetric uterine horn (UH) filled with amniotic fluid (AF).

Figure 15. Ultrasonographic image of a 28 day gestation in a female buffalo. The amniotic vesicle that contains the embryo is visible as well as the allantoic fluid. At this stage, it is possible to visualize the heartbeat.

In general, it is accepted that the fetal heartbeat can be detected by RUS between days 25-28 of gestation in buffaloes [5,92-94]. One of the more common mistakes in the diagnosis of early pregnancy can occur when interpreting the presence of non-echogenic fluids contained in the uterus as a positive sign of pregnancy [64]. Figure 14 shows an example of this situation where the examiner can see fluid contained in an organ; in this case it is the urinary bladder (UB). To avoid this type of confusion, it is recommended to perform a systematic examination of the uterine horn along its entire length with special emphasis on the ipsilateral uterine horn of the corpus luteum until the fetus can be visualized (Fig. 15). The ultrasonographic features of gestation in buffaloes have been published elsewhere [4,95].

The use of Doppler ultrasonography in animal reproduction research is more recent but not less important, and several studies have demonstrated the relationship of blood flow and ovarian and uterine function throughout the estrous cycle and pregnancy. Doppler ultrasonography allows the characterization and measurement of blood flow, and can be used to indirectly make inferences regarding the functionality of different organs. Doppler ultrasonography is a non-invasive real time pulse-wave technique used for the transrectal study of the reproductive system hemodynamics in large animals. This technique is based on the Doppler Effect principle that proposes the change in frequency of a wave for an observer (red blood cells) moving relative to the source of the respective wave (ultrasonic transducer). This method has shown to be effective and useful for the evaluation of the in vivo reproductive tract of different species increasing the diagnostic, monitoring, and predictive values of the RUS. However, an accurate and truthful ultrasonic examination requires the knowledge of the Doppler ultrasonography principles [2,36,38]. One application for buffaloes is the assessment of early embryo viability [96] (Fig. 16).

Figure 16. Color Doppler ultrasonography of a buffalo on Day 30 of gestation. The heart has been monitored and embryo viability has been assessed.

Reference data to assess function and perfusion of uteroplacental tissues by assessment of uterine blood flow parameters is sparsely available for buffaloes [97]. This could be a valuable tool to determine hemodynamic changes during pregnancy, ovulation and corpus luteum formation, and folliculogenesis among others. At some point, this technology will help veterinarians to diagnose some pathological conditions that affect the reproductive performance of buffalo species.

5.2. Assessing Fetal Development and Fetal Viability

Pregnancy loss contributes to reproductive inefficiency because fertility assessed at any point during pregnancy is a function of both conception rate and pregnancy loss [98]. As pregnancy progresses, it is possible to observe changes in fetal size and logically in the diameter of the pregnant horn. Fig. 17 shows a 38 Day old embryo scanned with a linear probe with a frequency of 7.0 MHz. The amniotic vesicle can be clearly visualized. It should be noted that the appearance of the uterine fluids in the course of a viable and healthy pregnancy must be non-echogenic and cell detritus-free.

Figure 17. Sonogram of a healthy buffalo gestation of 38 days. The amniotic vesicle, the uterine fluids and the fetus are shown.

Embryonic mortality in buffalo cows is considered one of the major causes of fertility loss, [18,19] and seems to be especially problematic in buffaloes that were not bred during their reproductive season. Prevalence of embryonic loss in animals bred by artificial insemination (AI) out of the favorable breeding season is typically 20-40% [18]. Also, in naturally mated buffaloes, the prevalence of embryonic death is about 20% with a higher frequency observed between 28-60 days of gestation [19,99]. RUS is a suitable tool to diagnose and assess embryo mortality in buffalo. Fig. 18 shows a Doppler ultrasound of a 35 day pregnant buffalo presented for pregnancy diagnosis. In addition to the low amount of fluid, the absence of a heartbeat was a confirmation of embryonic death. A previous study using color Doppler ultrasonography utilized the blood flow to the CL as a basis of predicting embryonic mortality [96]. Non-pregnant buffaloes on Day 25 after AI showed higher resistive index and pulsatility index values on CL compared to pregnant buffaloes [100].

Figure 18. Doppler ultrasound scan of the uterus of a 35 day pregnant buffalo. This is a positive diagnosis of early embryonic death. The reduced amount of fluid is evident, the amniotic vesicle is not observed, and the fetal heartbeat was absent. UH (Uterine horn).

There are many cases in which rectal examination yields a positive diagnosis of pregnancy based on signs such as membrane slip and palpation of the amniotic vesicle. However, it is sometimes difficult for the clinician to recognize a non–viable gestation if such signs are present during the examination (Fig. 19) and therefore, a false positive diagnosis would be arrived. RUS allows determining the viability of the gestation and the normal development of the fetus.

Figure 19. Ultrasonographic exam of a 53 day pregnant female buffalo. An empty amniotic vesicle is shown, also a slight change in the echogenicity of the amniotic fluid is observed. This animal showed positive signs of pregnancy, although the fetus was not viable.

RUS has shown to be a more sensitive method to evaluate fetal development. Other structures monitored by ultrasonographic evaluation are the fetus-placental union; the thickening of the uterine and fetal cotyledon (placentomes) can be observed by ultrasound around Day 45-50. However, they become palpable during the rectal examination after Day 75 of gestation. After Day 60 it is possible to recognize many fetal parts as well as the umbilical cord and placentomes, and potential changes in the quality of the allantoic and amniotic fluids can be observed (Fig. 20, Fig. 21, Fig. 22 and Fig. 23). A recent study [101] evaluated the appearance of combined thickness of the uterus and placenta (CTUP) from 2 month to full term gestation in buffalo that can be used to assess fetal development and function. The CTUP increased monthly from 2.5 mm at the 2nd month to reach 12 mm at full term.

Figure 20. A 60 day buffalo fetus. The thorax, ribs and lungs are observed as well as the umbilicus entrance into the fetal abdomen, and the fetal abomasum (stomach). In addition, the amniotic vesicle is also visible.

Estimation of fetal age, monitoring of fetal growth and development during gestation and diagnosis of pregnancy disorders can be performed by ultrasound. Ultrasonographic fetometry studies have been conducted in cows [65,88,102] and buffaloes [103].

Figure 21. Buffalo fetus of 120 days. The fetal heart beat rate could be measured at this time. The evaluation of the fetal thorax, uterine fluids, and placentome development, are important to assess fetal viability.

The gestational age prediction by ultrasonographic fetal measurements in buffalo could provide a relatively easy means to observe many events of pregnancy, either for research purposes or out of clinical interest during the investigation of pregnancy loss and abortion.

Figure 22. A. An 80 day old buffalo fetus, it is easy to observe the skull, eyeball orbit, the nostrils and the right front leg. Figure 22 B is showing a fetus of 150 days. The diameter of the orbit could be used to assess fetal age.

Currently RUS is available at a relative low cost, whereas the precision depends upon the clinical experience of the operator and the criteria for interpretation of the image. Advances in prenatal medicine and neonatology based on this tool are promissory to improve the diagnosis of many conditions that may affect fetal viability and development. One limitation could be the increased size of the fetus if the examination is performed by transrectal approach. Instead, it is possible to use a convex or sectorial transducer of 3.0-3.5 MHz to perform a trans-abdominal examination of the placenta and the fetus.

Figure 23. A. Ultrasound image of the umbilical cord at Day 150 of buffalo pregnancy. 23B. Ultrasonographic view of a 150 day pregnant buffalo. Arrows show placentomes of different sizes.

5.3. Fetal Gender Determination

This is one of the most attractive applications of the RUS, especially in farms that handle programs of intensive selection insemination, embryo transfers, or for commercial purposes of marketing of pregnant animals, where the gender of the calf is guaranteed. This technique, however, requires a higher performance since the reliability of the results depends on: age of the fetus, ability to locate and visualize the fetal structures, and type of equipment used. It is reported that the efficiency of the procedure can reach up to 97% if it is performed between 60-100 days of gestation [4,91].

The most accepted criterion to determine fetal sexing is the position of the genital tubercle (GT). The genital tubercle is the embryonic structure that will give origin to the penis and prepuce in the case of males and the vulva and clitoris in the case of the females (Fig. 24). Before Day 55 of gestation, the GT is located between the hind limbs in both sexes, however, the GT migrates towards the umbilicus in the male, and towards to the tail in the female between 40-60 days of gestation [64,65].

Figure 24. Anatomical site of the genital tubercle. View of the location of the genital tubercle in the male near the umbilicus (Panel A) and in the female under the tail (Panel B). Genital Tubercle (GT), Umbilical cord (UC). Adapted from Ginther (2004) [64].

There are three important anatomical reference points to achieve an efficient gender determination; together these allow one to determine the position of the fetus: 1) the fetal head, 2) the heartbeat, and 3) the umbilical cord. These 3 structures are much easier to recognize than the rear and hind limbs when it comes to know what the position of the fetus is. It is advisable to perform a good retraction of the uterine horn in order to facilitate both the handling and the ultrasound examination [104].

Once the fetus is located and its position is defined, it is possible to obtain different images depending on the relationship between the fetus and the position of the transducer: longitudinal (dorsal or ventral), lateral, or cross-sectional. The technique consists of scanning the fetus with the transducer using very slow movements to obtain better results, from the skull in caudal direction to identify the chest, the heartbeat, the abdominal cavity, the umbilical region and caudally, the perineal region [105,106]. Frequencies from 5.0 to 8.0 MHz can be used for gender determination in bubaline fetuses [94,95] as well as in other species [104]. It is always advisable to use low frequencies since they allow observing the fetus in its entire length which hastens the diagnosis. Ultrasound scanners that allow a change in the frequency using the same transducer, permit the combination of these two options making the diagnosis more efficient. Once the fetus is adequately located, attention should be focused to the area of the umbilical cord. In the case of males, the genital tubercle can be recognized as a hyper-echogenic structure immediately behind the umbilical cord at the point of entry into the fetal abdomen (Fig. 25Aand Fig. 25B).

Figure 25. A. Ultrasonographic image of a 68-day male buffalo fetus. The umbilicus is entering into the fetal abdomen. The hyper-echogenic structure visualized behind the umbilicus corresponds to the genital tubercle. The rear right leg and the fetal pelvic bones are also shown. Figure 25 B. Ultrasound of a 90-day male buffalo fetus, the umbilical cord, amnion, a placentome and the echogenic image of the prepuce in a male fetus are shown.

In the case of females, the genital tubercle can be observed in the caudal region near the root of the tail. It is necessary to differentiate between the image of the genital tubercle and the coccygeal vertebrae. It is important to emphasize that if the genital tubercle is not observed in the umbilical region, it does not necessarily mean that it is a female fetus. The clinician must complete the examination of the fetus to establish the presence or absence of the genital tubercle in the coccygeal region. In either case, if the genital tubercle is visualized near the tail, the examination must be repeated at a later date to confirm the diagnosis.

In Figure 26A, the fetal head can be located as well as the front legs (FL), the rear legs (RL) and the umbilical cord (U). At this point, the image in Figure 26A does not show the genital tubercle behind the tail suggesting a male diagnosis; however, it is mandatory to visualize the GT for the diagnosis to be accurate. Figure 26 B shows the same fetus in a better position that allows to clearly observe the image of GT in the caudal region confirming the diagnosis of a female fetus. Figure 26C shows an image of a female fetus at Day 90 of gestation. Here it is possible to show the difference between the image obtained from the coccygeal vertebrae at the tail (T) and the genital tubercle. In buffaloes the best window for fetal sexing using transrectal ultrasonography was found between the 10th and 18th week of gestation [13] with an overall accuracy of 64% to 97% [13,14].

Figure 26. A. Reproductive ultrasound of a bubaline fetus for gender determination by Day 60 of gestation. 26 B and 26 C. Female fetus at Day 60 and 90 of gestation. Rear leg (RL), front Leg (FL), Genital Tuberculum (GT), Umbilicus (U), Tail (T).

Figure 27. A. Ultrasonographic image of a 68 day female fetus. The genital tubercle (GT) is visible in a cross-sectional scan of the fetus at the level of the tail as a hyperechoic structure. Figure 27 B. Ultrasound of a 90 day female fetus, the amnion, and a set of placentomes are shown at the top. The echogenic image of the fetal vulva can be observed under the fetal tail.