CHAPTER 89 Pregnancy Diagnosis
Pregnancy diagnosis in sheep is an important management practice because of the impact of reproductive performance on economic return from the flock. Early pregnancy diagnosis, determination of fetal numbers and estimation of day of gestation, and recognition of abnormalities of pregnancy can provide producers with significant management opportunities to enhance reproductive efficiency. Historically, producers of small ruminants have had few options with regard to pregnancy diagnosis. They have been forced to wait a sufficient period of time to visibly detect return to estrus or to detect pregnancy based on mammary development and abdominal enlargement. The latter method, although relatively accurate in multiparous females, can give false negative results in primiparous females and can only be performed with a high degree of accuracy during the latter stages of gestation. Earlier diagnosis based on return to estrus following mating has commonly relied on use of a vasectomized ram with brisket paint or fitted with a crayon marking harness. The nonpregnant ewes that continue to cycle during the breeding season are marked by the teaser ram. However, some pregnant ewes will continue to exhibit estrus and be marked by the teaser ram. High-libido teaser rams may mark pregnant ewes that are not exhibiting estrus, as well as nonpregnant ewes not exhibiting estrus.
This technique has been used effectively for rapid testing of up to 400 to 600 ewes per day for pregnancy diagno sis and determination of number of fetuses; however, the cost of the equipment and risk to operator and animal health makes radiography impractical.
This technique requires a lubricated glass rod (1.5 by 50 cm) to be inserted into the rectum of ewes lying on their back. With the operator’s hand on the abdomen and movement of the rod with the other hand, the attempt to diagnose pregnancy was done without a high level of accuracy until late in gestation. Although cheap, simple, and quick, this technique has too low a rate of accuracy to be useful.
Determination of the concentration of progesterone in blood using an enzyme immunoassay provides a reliable method for detecting pregnancy as concentrations are expected to be high for pregnant ewes that maintain functional corpora lutea. However, this method is not reliable for diagnosing the absence of pregnancy due to embryonic death, uterine pathology, or failure of the corpus luteum to regress at the expected time. This method can allow one to estimate the presence of multiple fetuses as the increase in number of corpora lutea is associated with higher concentrations of progesterone in blood, but one cannot accurately estimate day of gestation or the exact number of fetuses.
This method has not proved to be useful because it is most accurate from after day 70 of gestation and does not provide useful information with respect to number of fetuses or estimates of day of gestation.
Pregnancy-specific protein B (PSPB) from binucleate trophectoderm cells of conceptuses of ruminants is detectable in blood by about days 18 to 19 after onset of estrus. PSPB assays have been reported to provide 100% accuracy in detecting pregnancy, but only 83% accuracy in detecting nonpregnancy between days 26 and 106 of gestation.
Interferon tau is the pregnancy recognition signal in ruminants that acts to inhibit expression of estrogen and oxytocin receptors by uterine epithelia to prevent their release of luteolytic pulses of prostaglandin F2 alpha (PGF2α). Many unsuccessful attempts have been made to detect interferon tau in circulating blood and lymphatic vessels. However, there are reports of increases in expression of some interferon-stimulated genes in immune cells in blood of sheep (Mx protein) and cattle (2′,5′-oligoadenylate synthase). The use of these interferon-stimulated gene products has not been proved reliable as an indication of pregnancy because any type I interferon, for example, as a result of infection or other pathology, may cause increases in expression of these interferon-stimulated genes. With respect to pregnancy diagnosis, the risk for false positives is expected to be high.
The inability of traditional and historical diagnostic methods to provide early, accurate pregnancy information has led to the development of alternate techniques. During the past 20 years, various types of ultrasonographic systems have been developed to diagnose pregnancy in small ruminants. Ultrasound can be defined as high-frequency sound waves above the audible range. The frequency of these sound waves is described in cycles per second, or hertz (Hz). Frequencies in the range of 20 to 20,000 Hz can be detected by the human ear; however, frequencies used for diagnostic ultrasonography are typically in the range of 2 to 10 million cycles per second, or megahertz (MHz). Two basic types of ultrasonography have been used for pregnancy diagnosis, amplitude depth (A-mode) and brightness (B-mode).
A-mode systems are based on two different principles: sonar and Doppler shift. A-mode systems that utilize the sonar principle rely on echoes to detect differences in impedance between tissue interfaces that are separated by fluid. Accuracy is in the 80% to 85% range from day 60 of gestation to term. A-mode systems that utilize the Doppler shift principle produce a signal (either light or audible sound) when sound waves rebound from a vibrating surface, such as the maternal middle uterine artery or the fetal heart. The frequency of the waves reflected from the vibrating surface is different from that transmitted. Although Doppler systems should identify multiple fetuses by detecting two or more fetal heartbeats, actual results in practice have been poor. Depending on operator experience, stage of gestation, and animal preparation, the accuracy of the Doppler systems ranges from 70% to 90%.
In recent years, B-mode or real-time ultrasonography has become the preferred means of pregnancy diagnosis for small ruminants. Real-time ultrasonography gets its name from the appearance of image movement. The impression of movement is given by the rapid change of the image as it is displayed and is analogous to an animated cartoon. In addition to determining pregnancy status, the image of the gravid uterus produced by real-time ultrasonography allows the operator to count fetuses, determine stage of gestation, and evaluate the health status of the pregnancy. Competent operators can provide this information rapidly (250 to 300 ewes per hour) with 95% to 98% accuracy.
Real-time ultrasonographic systems include a handheld transducer and an electronic control unit with a cathode ray tube (CRT) monitor. An electrical current passes through one or more piezoelectric crystals con tained in the transducer and is converted into short (1 μsec) bursts of sound. Between bursts, the transducer acts as a receiver and converts the returning ultrasound waves into electrical impulses that produce a two-dimensional image on the CRT monitor. The brightness of the image depends on strength of the returning signal, which is determined by both the position and the density of the reflecting tissues.
Intrarectal and transabdominal placements of the probe for ultrasonographic scanning are used for pregnancy diagnosis in small ruminants. For transabdominal scans, the transducer is placed in contact with the area of the abdomen in the inguinal region next to the udder that lacks wool. Ultrasound waves will not pass through air; therefore, a coupling agent (such as carboxymethylcellulose) is necessary to ensure proper contact between the transducer and tissue. Wool, dirt, grease, and feces disperse sound waves, resulting in an inferior or nonreadable image.
Pregnancy diagnosis in small ruminants typically utilizes ultrasound frequencies from 3.5 to 7.5 MHz. The lower frequencies provide deeper penetration into underlying tissues but poorer resolution, and higher frequencies do not penetrate as deeply, but provide greater definition of superficial structures. A 5-MHz transducer is most versatile and provides a good diagnostic image for either transabdominal or intrarectal scanning. A 3.5-MHz transducer works well for transabdominal scans, but a 7.5-MHz transducer is preferred for transrectal diagnosis of early pregnancy and examination of ovarian structures.
Linear array transducers tend to be more versatile for veterinary practitioners who use ultrasonography in a variety of species and for multiple applications. For pregnancy diagnosis, the physical size and shape of the linear transducer lends itself to both intrarectal scans of the abdomen and transabdominal scans. The linear array transducer contains a number of piezoelectric crystals arranged in a line to emit sound (“fire”) sequentially and then receive the rebounded sound waves to produce a rectangular image.
Intrarectal scans provide excellent resolution and definition of fetal and placental structures and can be used to diagnose pregnancy from as early as day 18 to as late as day 120 of pregnancy. However, it should be cautioned that very early pregnancy diagnosis and fetal counts prior to day 45 of gestation may not correlate well with lambing rates, as ewes are at greater risk of fetal loss during the early stages of gestation.
The accuracy of counting fetuses using rectal scans is limited because the entire uterus cannot be examined and the operator may experience difficulty in maintaining orientation for accurate counting of fetuses. As pregnancy advances beyond day 60, accuracy in counting fetuses increases, but as the gravid uterus moves further ventral and cranial with advancing gestation, it becomes more difficult to advance the probe sufficiently to scan the entire uterus.
With the ewe restrained in dorsal recumbency, rectal scanning using a 7.5-MHz transducer is an excellent means for examining ovarian structures (corpora lutea and follicles), and for diagnosis of very early pregnancy. In this position, the reproductive tract lies against the transducer, allowing one to obtain a complete image of the uterus and ovaries.
Linear array transducers can also be used effectively transabdominally to diagnose pregnancy between days 35 and 100 or more of gestation. Operators can determine pregnancy status and fetal number, but accurate fetal counts require patience and experience. The entire uterus usually cannot be imaged with a single placement of the transducer, so the operator must remember fetal orientation to ensure that the same fetus is not counted twice.