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An update on the use of B-mode ultrasonography in female pig reproduction
Theriogenology 67 (2007) 901–911 www.theriojournal.com
Review
An update on the use of B-mode ultrasonography in female pig reproduction J. Kauffold a,*, G.C. Althouse b a
Large Animal Clinic for Theriogenology and Ambulatory Services, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 29, 04103 Leipzig, Germany b School of Veterinary Medicine, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348, USA Received 10 May 2006; received in revised form 23 October 2006; accepted 17 December 2006
Abstract After technological advances allowed for the adaptation of B-mode ultrasonography equipment for use in pig facilities, ultrasonography quickly established itself as an ideal diagnostic aid for determining pregnancy status in pigs. In recent years, Bmode ultrasonography has found increased application in its use for monitoring ovarian activity and in estimating time of ovulation in pigs. B-mode ultrasonography is also valuable in providing a detailed assessment of the sow’s ovaries and uterus to determine if pathological conditions exist, which could be contributing to poor individual or herd reproductive performance. In its most recent application in pigs, the gilt genital tract has been characterized peripubertally by ultrasonography in order to detect onset of puberty. The purpose of this review is to provide an update on the current status of B-mode ultrasonography in pig reproduction, and how this technology can be of value when used in pig production medicine. # 2007 Elsevier Inc. All rights reserved. Keywords: Pig; Ovary; Uterus; Puberty; B-mode ultrasonography
Contents 1. 2. 3.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical requirements, use and equipment biosecurity . . . . . . . . . . . . . . . . . . . . Ultrasonographic examination of the female genital tract . . . . . . . . . . . . . . . . . . . 3.1. Ultrasonographic examination of the gravid female and pregnancy failure. . . 3.2. Ultrasonographic examination to monitor follicular dynamics and ovulation . 3.3. Ultrasonographic examination of the gilt and determination of puberty status 3.4. Ultrasonographic examination of the non-productive female . . . . . . . . . . . . Incorporation of ultrasonography of the female genital tract in pig production . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +49 3419738364; fax: +49 3419738398. E-mail address: [email protected] (J. Kauffold). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.12.005
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1. Introduction For many years ultrasonography has proven itself an excellent diagnostic tool for the gynaecological examination in many farm animal species [1,2]. In 1983, Inaba et al. [3] was one of the first who demonstrated the value of B-mode ultrasonography in diagnosing pregnancy in pigs. In the following years, multiple studies were performed [e.g., 4–12] assessing the use of B-mode ultrasonography in pigs. B-mode ultrasonography has repeatedly proven its superiority to other methods of pregnancy diagnosis such as service non-return rates [13,14] and endocrinological testing modalities [15,16]. Altogether, the pig industry has embraced this technology for diagnosing pregnancy in pig production operations [8,17], however, there still exist issues of dispute such as the day of first use postbreeding or the necessity for serial examinations on individual animals. In 1985, Botero et al. [4] reported the successful use of ultrasonography in pigs for the detection of ovarian cysts. It took a further 4 years until a research group demonstrated the use of transcutaneous B-mode ultrasonography in visualizing normal, peri-ovulatory ovarian structures and to monitor the ovulation process [18]. Further work corroborated the value of B-mode ultrasonography for diagnosing ovarian pathology [19– 21]. These applications have had a favourable impact both in their use in scientific studies [22–26], and much less so in pig production systems [9,17,27,28]. In contrast to its use in visualization of the ovary, only a few studies have applied B-mode ultrasonography in the assessment of the porcine uterus [4,7,11]. This is somewhat surprising given that uterine disorders account for a large percentage of reproductive failure in pigs [29,30]. Ultrasonography has been promoted in the assessment of intrauterine fluid, other than pregnancy, as an indicator of uterine inflammation and disease [7,17], and has shown value in providing detailed information on the overall health of the porcine uterus [31–33]. Eight years back Martinat-Botte´ et al. [34] presented a condensed text on ultrasonography in pig reproduction. Since then, there has been a large amount of new information generated on the topic. The purpose of this review is to summarize the current knowledge on the use of ultrasonography in assessing the reproductive tract in pigs. Along with reviewing its use in pregnancy diagnosis, special emphasis will be placed on new information garnered from recent investigations studying the use of B-mode ultrasonography in puberty [35,36] and on uterine pathology diagnosis [31,37].
Inferences to the value of this diagnostic aid in pig production and practice will be made. 2. Technical requirements, use and equipment biosecurity Several transducers have been used for gynaecological examination of the female pig, including lineararray [8,14,38] and convex-array transducers [17,39], as well as mechanical sectors [4,9,15]. Each transducer has particular advantages and disadvantages [10,17,40], with no one transducer clearly delineated as optimal for use in pigs. From the authors’ experience, most commercially available transducers have sufficient resolution and durability for use in pig practice, with personal preferences usually dictating which type is used. To date, there have been no reports in the scientific literature, which address specific requirements for ultrasound machines used in pig practice. According to the few available reports [17,40] and the authors’ experience, machines should be portable (i.e., lightweight and having an internal power supply) and robust. Portability is frequently regarded as a primary factor in machine choice given the mobility needed by personnel in using this equipment in group-housed or crated sows. For assessment of the sow reproductive tract, a transducer frequency of 5.0 MHz provides the most versatility [8,9,11,31–33]. Other frequencies, including 3.5 and 7.0 MHz, have been investigated for use in pregnancy diagnosis and ovary examination [3– 5,10,17]. Transabdominal/transcutaneous probe placement and scanning is commonly used in the industry for early, accurate diagnosis of pregnancy [6,11,38]. Transrectal ultrasonography has also been advocated for this purpose [40] and, in some aspects preferred [6,17]. In particular, some authors [17,22–25,28,41] favour transrectal scanning for assessing the ovaries. Comparison of transcutaneous and transrectal procedures was performed in the early 1990’s [41]. It was found that transcutaneous assessment was at a disadvantage for a number of reasons including difficulty in obtaining clear images due to interference with intestinal tissues, ambiguous detection and counting of corpora lutea, and the technical experience needed to perform the procedure properly. Moreover, repeated transrectal scanning has been found to not have an effect on reproductive outcomes such as fertilization and embryonic development [42]. Utilizing currently available equipment, the authors have successfully used transcutaneous scanning to detect puberty in gilts [36] and to characterize the ovaries [43] and uteri [31] in reproductively failed pigs. This route of scanning was
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found to be performed relatively simple, fast and safe procedure (e.g., due to no necessity to remove faeces [12] and of no risk of trauma to the animals [39,43]). Although ultrasound machines used in pig production operations have been identified as a biosecurity risk [16,17], only one recent study has addressed this issue [44]. According to this study [44], ultrasound equipment used in pig units was found to harbour several bacterial species, including the potentially pathogenic Streptococcus sp. Of higher interest, porcine reproductive and respiratory viral RNA was able to be recovered from the inside of different types of ultrasound machines. Given these findings, ultrasound equipment may carry a risk of being a fomite for the transmission of diseases. It is recommended that machines and all equipment be cleaned and disinfected after each use. While on the farm, equipment should be covered to aid in biosecurity, and transport of equipment between farms should be minimized. If travel between farms is necessary, sufficient sanitation protocols, including equipment downtime, should be used. 3. Ultrasonographic examination of the female genital tract 3.1. Ultrasonographic examination of the gravid female and pregnancy failure The earliest detection of pregnancy-specific structures (i.e., fluid-filled embryonic vesicles) has been reported to vary between 12 and 16 days of pregnancy [6,11,17,38]. Early pregnancy detection at 12–16 days can be, however, challenging in the sow using current transcutaneous ultrasonography techniques. As with other species, pregnancy detection rates in pigs improve as pregnancy progresses [8,11,17] and the conceptus increases in size [8,11]. While the developing conceptus has a 4.0 mm transverse diameter on day 15 of gestation, by days 18–22 the conceptus measures approximately 10 mm in diameter [11]. Using transrectal scanning and a 7.5 MHz probe, the embryo can usually be first detected on day 18 [12]. With transcutaneous scanning and a 5.0 MHz probe, pregnancy can be determined between 19 and 20 days of gestation [6,8]. Heartbeats appear between days 21 [6] and 25 [38], and are appropriate to determine viability of embryos or fetuses [9]. Transcutaneous Doppler cardiography has been used to study fetal heart rate (FHR) of eight fetuses in the last 10 days of gestation [45]. FHR decreased in seven normal fetuses toward parturition, but increased in a fetus with abdominal ascites [45] suggesting that antepartum Doppler FHR
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measurement might be feasible to predict viability of fetal and newborn pigs [46]. Both the fetal eye orbit and the stomach have been first detected on day 49 of gestation by transcutaneous scanning using a frequency of 5.0 MHz [6]. The diameter of the fetal heart, eye orbits and stomach grow at a linear rate (r = 0.75–0.92), and estimation of their transverse or longitudinal dimension can be helpful in determining gestational stage if breeding dates are unknown [6]. In pregnant cattle, endometrial echogenicity (expressed as the mean grey level) declined between days 7 and 13 after ovulation, followed by a subsequent moderate rise reaching medium values on day 15 [47]. These same changes in endometrial echogenicity were not present in cyclic cows [48,49]. Similarly, a decline in echogenicity of the cross-sectional image of the uterine horn was observed in pregnant gilts between days 12 and 14 of gestation, but was absent in nongravid, control gilts (Fig. 1; J. Kauffold, B. von dem Bussche, A. Sobiraj, M. Wendt; unpublished observation). This decline in echogenicity might be due, at least in part, to an increased endometrial oedema, as only uterine cross-sections being completely devoid of luminal fluid were measured. Preliminary work by the authors suggests that this temporal change in endometrial echogenicity might have value in the indirect diagnosis of early pregnancy and embryonic mortality. However, the level of training and necessary equipment resolution may preclude its use as a routine means for very early pregnancy diagnosis in pigs.
Fig. 1. Echogenicity of uterine horn cross-sections expressed as the mean grey level S.D. in pregnant (squares; n = 4) and non-pregnant (circles; n = 8) gilts on days 5–16 post-ovulation (day 0). Echogenicity was estimated using grey scale analysis (grey levels ranged from 0 to 32) with a HS 2000 (HONDA Electronics, Tokyo, Japan), a 5.0 MHz linear-array transducer and transcutaneous scanning. For analysis, a mean of 6.4 entire uterine cross-sections per gilt were estimated using standardized equipment settings. Pregnant females had a significantly (P < 0.05) lower echogenicity between days 12 and 14 than the nonpregnant females (values with letters (a and b) differ; J. Kauffold, B. von dem Bussche, A. Sobiraj, M. Wendt; unpublished observation).
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Successful diagnosis of pregnancy status in early gestation depends upon transducer frequency, route of application, technical expertise, experience and other factors inherent to the patient themselves [9,17]. For instance, correct diagnosis of pregnancy status was higher on day 21 if a 5.0 MHz transducer was used transcutaneously versus a 3.5 MHz transducer [9,10]. A transrectal approach allowed earlier pregnancy diagnosis with greater accuracy than examinations done by the transcutaneous procedure [6]. In general, recognition of pregnancy is easier than non-pregnancy [8,10]. While pregnancy-specific structures definitely indicate pregnancy, their absence does not necessarily indicate a non-gravid status, as in some pregnant females, particularly during early stages, structures indicative of pregnancy can remain undetected [6]. Pregnancy diagnosis can approach 100% sensitivity as early as day(s) 19 [11], 20–21 [8] or 21–23 [9] of gestation. Specificity (i.e., ability to distinguish from a non-gravid sow), however, was almost always significantly lower, irrespective of the stage of gestation [6–9]. The main reason for the low specificity is that females initially identified as pregnant by ultrasonography and later returned to estrus or failed to farrow were presumed to be false positives [8,10,11,37]. This presumption, however, can be erroneous as those females identified as pregnant at the time of initial examination may have indeed lost pregnancy thereafter [8,10–12,37]. Thus, ultrasonography is maybe much more accurate in the
diagnosis of non-pregnant animals than the available data suggests [8,37]. The necessity of re-confirming pregnancy at a later date in females initially diagnosed early in gestation is a current area of debate [7,8,11,37]. This is in lieu of the fact that the rate of embryonic mortality in sows is highest up until day 30 of gestation [50,51]. Recent work, however, suggests that re-confirming pregnancy on a routine basis may be of limited value given that the number of females diagnosed pregnant and later farrowed were found to be non-significantly different [37]. Ultrasonography has clinical value for diagnosing pregnancy failure and early embryonic death leading to either fetal mummification [12] or partial embryonic decomposition (Fig. 2A; [8]). Ultrasonography can be valuable in assessing females which had recently aborted or had undergone complete embryonic loss based upon the echogenicity and non-typical shape of the fluid-filled uterine structures devoid of the embryo proper or enlarged uteri exhibiting extreme heterogeneous echotexture [37]. Diagnosis is aided from additional information such as data on breeding and/or clinical signs observed. Another, but rather uncommon diagnosis is hydrometra, which has been observed twice among approximately 3 105animals scanned by us for pregnancy during the last 12 years. In both cases of hydrometra, the uterus was enlarged with thinning of the uterine wall through extreme intrauterine accumulation of fluid (Fig. 2B).
Fig. 2. Images obtained by transcutaneous ultrasonography using a HS120 ultrasound machine (HONDA Electronics, Tokyo, Japan) and a 5.0 MHz linear-array transducer. (A) Decomposing embryo (arrow; taken from [8]). Note the loss in homogeneous echotexture and mottled appearance. (B) Hydrometra in a sow. The uterus is filled with large amounts of fluid, which has lead to an extreme thinning of the uterine wall (walls of two adjacent uterine horn cross-sections are delineated between arrows). Scale bar on the top and left margin in 0.5 cm.
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3.2. Ultrasonographic examination to monitor follicular dynamics and ovulation From recent reviews [52–55], ultrasonography has greatly increased our understanding of follicular dynamics in pigs. Most research has been done in the lactating pre-weaned and in the post-weaned sow. According to Lucy et al. [52], pre- and post-weaning follicular growth displays a wave-like pattern. This wave consists of a cohort of 20–30 follicles of 2 mm in size that synchronously starts to grow in response to FSH stimulation. During lactation, co-dominant follicles reach up to 5 mm and then regress. Post-weaning, those follicles appear to contribute to the pre-ovulatory pool [52]. Developmental stage of follicles at weaning appears to influence weaning-to-estrus interval (WEI), as females having follicles being in the developmental stage of a cohort come into estrus earlier than females having follicles of similar size but weaned when the follicular cohort was regressing [50]. The size of the follicles at weaning seems to be equally important, as a reduced follicle size on day 2 or 3 after weaning was associated with an increased WEI and weaning-toovulation interval [24,56]. Support for those observations also comes from a recent study [57] showing that sows treated pre-weaning with multiple injections of charcoal-extracted porcine follicular fluid, aimed to suppress FSH and follicular growth, had a mean follicle size at weaning smaller than that of saline-treated controls (1 versus 3 mm) and a longer WEI (6.1 0.4 versus 4.7 0.4). The relationship, however, does not appear to be strong enough to be used to predict estrus or ovulation on the basis of follicle size determined around weaning [24,56]. Through ultrasonography a number of factors have been identified in association with reduced follicle size peri-weaning, including high ambient temperature [52] and low energy in feed [58] during lactation, first parity and bad body condition [24]. Once the post-weaning follicular growth has emerged, the dynamics of follicular development (i.e., the slope) appear to be unaffected by follicular size around weaning [24,52]. At the beginning of estrus [23,28] and 20 h thereafter [56], pre-ovulatory follicles average between 7 and 9 mm [56], but size can vary considerably between females. Soede et al. [56] observed that follicles measuring <7 mm 16 h prior to ovulation increased in size up to ovulation, while follicles >7 mm do not change or become somewhat smaller. Pre-ovulatory follicle size does not appear to affect reproductive traits, as no association was found between follicle size and WEI, fertilization rate, as well as embryo development and diversity [56]. Minor
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reduction in size and changes from a spherical to a more ovoid [52,56] or longish shape [57] of the pre-ovulatory follicles immediately preceding ovulation have been reported, with a suggested loss in follicle turgidity [56,59], similar to that observed in mares [60]. These changes have been proposed to be candidate parameters to predict ovulation time in pigs [59]. However, there appears to be no reports in the scientific literature to support this. Considering the importance follicular turgidity plays in determining ovulation time in mares [60], it might be worthy of further investigation in pigs. Initiation of ovulation and the inter-ovulatory period has been estimated using sequential examinations of the ovaries to follow a reduction in the number of follicles [17,28]. The ovulation process can take up to several hours [22,61], and pre-ovulatory follicles can be observed concomitant with corpora haemorrhagica [43]; the latter are clearly identified by characteristic echogenic appearance [43]. Ovulation was considered to be completed if sequential examinations showed the disappearance of all pre-ovulatory follicles [17,21,39] or a marked reduction in the number of pre-ovulatory follicles relative to previous observations [28], or the ovary could not be visualized [62]. Failure to visualize the ovary, however, seems to be a rather unreliable criterion given that in some situations the ovaries can not be located due to circumstances not related to disappearance of follicles [43]. The presence of corpora haemorrhagica while pre-ovulatory follicles are absent seems to be most indicative for ovulation completion [31,36,43]. Different intervals between examinations, ranging from 4 to 24 h, have been chosen for monitoring ovulation in pigs [28,56]. Although the closer the intervals are logically more precise in the diagnosis of ovulation time [28], the longer interval of 24 h is more desirable from a practical standpoint, and seems to be adequate when used appropriately [23,28]. A number of authors have emphasized that implementation of ultrasonography for monitoring ovulation in pig production would be economically beneficial (e.g., proper timing of ovulation, insemination timing, avoiding post-ovulatory inseminations, etc. [9,17,27,40,62]). Although the inherent difficulty in routine application of this technique in the field has precluded its widespread implementation so far, examples in the literature suggest that its application is increasing in practice [27,28,40]. The greatest value of monitoring ovulation in a farm setting is certainly to improve insemination strategy [9,28,40,62]. Almond [9] speculated that producers might be able to perform ultrasonography to monitor ovulation and might then inseminate sows individually.
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Fig. 3. Images obtained by transcutaneous ultrasonography using a HS120 ultrasound machine (HONDA Electronics, Tokyo, Japan) and a 5.0 MHz linear-array transducer. (A) Polycystic ovarian degeneration (POD): multiple, approximately 2.5 cm measuring anechogenic spherical structures without accompanying corpora lutea (CL). (B) POD, with cysts having a 3 mm thick wall (delineated between arrows) suggestive of luteinized tissue. (C) A 2.5 cm large ‘‘blood cyst’’ having similar echogenicity as a CL (refer to image D), but with an irregularly shaped small anechogenic cavum. Insert shows the same ovary post-mortem. (D) Oligocystic ovarian degeneration: two cysts (black arrows) accompanied by two clearly
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3.3. Ultrasonographic examination of the gilt and determination of puberty status Ultrasonography has been proven an appropriate tool for the diagnosis of puberty status in gilts. Both the evaluation of the ovaries and the uterus have been found suitable in determining gilts as either prepubertal or pubertal. Follicles of 2–5 mm in size and absence of any luteal structures in the ovary is a typical finding for prepubertal females [17,36]. Uterine size may also be of value in determining gilt sexual maturity. MartinatBotte´ et al. [35] collected uterine horn vertical diameters and employed an integrated computer program to calculate the ‘‘uterine area’’, i.e., an area covering all visible parts of the uterus within an ultrasound image. They estimated a sensitivity and specificity of 98.8 and 100%, respectively, for the diagnosis of prepubertal and pubertal gilt status. Kauffold et al. [36] also utilized uterine size in determining pubertal status, but measuring two or more cross-sections of the uterine horns in their maximum and minimum dimensions. Cross-sectional areas were calculated using the mathematical equation for an elliptical figure, and averaged all areas to give the mean sectional area of the uterine horns for a female. Accordingly, prepubertal females were found to have a cross-sectional area of 1 cm2, with pubertal animals exhibiting cross-sectional areas of 1.2 cm2. These determination values were then applied in a field study and results verified on the basis of progesterone blood concentrations and estrous behaviour. Accuracy of pubertal and prepubertal diagnosis was 91.0 and 100%, respectively. 3.4. Ultrasonographic examination of the nonproductive female During recent years, progress has been made in the use of ultrasonography to examine the genital tract of non-productive sows in order to detect reproductive disorders [17,34] and, more specifically, ovarian disorders and the diseased non-gravid uterus. Recognition of ovarian disorders in pigs is possible through the fact that physiological ovarian structures are frequently well-defined by ultrasonography [17,42,63]. Diagnosis of cystic ovarian disease is relatively simple [17,19,27,43], but complete delineation of ovarian cyst type via the ultrasonographic image can vary somewhat.
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Ovarian cysts are defined as fluid-filled structures that exceed a diameter of 11 [27] or 12 mm [28,64], as unovulated or luteinized follicles of 12–50 mm appearing large, smooth, circular and anechogenic [17], or as anechogenic structures with smooth, thin walls of 20 mm in diameter being present at least for 5 days after ovulation [21]. Follicular cysts, originating from unovulated follicles, usually appear as fluid-filled bubbles surrounded by a thin wall (Fig. 3A; [17,19,27,43,59]). In contrast, luteal cysts, originating from a developed corpora lutea, have a rather thick wall and a distinct lumen (Fig. 3B), which sometimes contains trabeculae-like structures [20], and are thus rather dissimilar to so called ‘‘blood cysts’’ (Fig. 3C) assumed to develop from overgrown corpora haemorrhagica [27,65]. The origin of luteinized cysts, if diagnosed, is impossible to determine since follicular cysts are also known to luteinize spontaneously [66,67]. Information on the cyst origin, however, is of lesser interest than is the observation of a luteinized wall per se, as it might indicate an ongoing regression process [21,43,67] and/or susceptibility of exogenous PGF2a administration. Besides cyst type, ovarian cysts can be distinguished on the basis of their number, as both single [19,43] and multiple cysts (i.e., 2, also referred as to oligocystic ovarian degeneration when accompanied by corpora lutea; Fig. 3D; [19,21,43]) have been described. Using ultrasonography, those conditions were estimated to occur between 7.6% [43] and approximately 30% [27] in breeding pigs and occasionally also observed preweaning [52], and consequences on reproductive performance such as reduction in litter size [27], lower farrowing rate [21,27] and higher frequency of returns to estrus [21] have been reported. Polycystic ovarian degeneration [17,19,21,43], also referred as to cystic follicular degeneration [27], is characterized by multiple fluid-filled bubbles (i.e., cysts) and the absence of corpora lutea [43]. This condition has been estimated to occur between 0.6 [27] and 4.3% [43] in breeding pigs on the basis of ultrasonographic examinations. It normally blocks reproductive function life-long and is a valid reason to cull females immediately. Individual sow cases of spontaneous regression, albeit after several weeks, have however been reported [66]. Another ovarian malfunction is ovulation failure. Pre-weaning ovulation was observed by Lucy et al. [52] in a small percentage of sows and certainly affects
recognizable CL (white arrows). (E) A para-ovarian cyst (arrow) of irregular shape and approximately 2 cm in diameter attached to an ovary having several large, pre-ovulatory follicles. (F) Intra-abdominal testicle-resembling structure found in a gilt. The structure is completely homogeneous in echogenicity, typical of intact testicles in the male. Insert shows the structure post-mortem. Scale bar on the top and left margin in 0.5 cm.
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expression of fertile estrus post-weaning. Delayed ovulation, defined as a follicle(s) that ovulate(s) 7 h after the majority of follicles has ovulated [19,27], has been observed in 3.8% out of 346 females using repeated ultrasonographic examination [19]. This condition, however, does not appear to affect reproductive performance [19,27]. Persistence of single medium sized follicles distinct from ovarian cysts beyond ovulation has been reported to occur occasionally in sows [22]. Complete ovulation failure has been diagnosed in weaned sows detected in standing estrus [23], and was associated with a short lactation length of 16 days [28]. Follicular size was inconsistent, as those sows could have variable sized follicles ranging from small to medium [28] or of pre-ovulatory size [23]. Whether inactive ovaries and corpora lutea (pseudo)persistent (CLP) are primarily ovarian malfunctions is a matter of question. Inactive ovaries, characterized by several follicles of predominantly smaller size, were one major finding in non-pregnant first service sows and gilts scanned between days 20 and 114 after breeding [43], and those females usually reported being anestrous for a longer interval [32,43]. Failed follicle growth has been repeatedly observed by ultrasonography in sows after weaning. Insufficient gonadotropin support (i.e., LH), necessary for final follicular maturation, was suggested as the main reason for failure [23,28,52]. Among those sows experiencing failed follicular growth, two distinct populations have been described, one without any post-weaning follicle growth, and the other with follicle growth up to 4 or 5 mm without further progression to a pre-ovulatory size [23,28,52,68]. CLP might be the result of embryonic mortality [23] or exposure to zearalenone [69,70] and do not differ from cyclic corpora lutea in the ultasonographic appearance [23,32]. Differentiation might only be possible if additional information (such as on last estrus/breeding) is available and pregnancy definitely excluded. Para-ovarian cysts have been detected by ultrasonography in 18 of 346 females scanned [19,28]. They are of different size (>1 cm up to several cm), completely anechogenic, but variable in shape (round, ovoid, polygonal; Fig. 3E). They do not appear to impair reproduction function and can easily be source for misdiagnosis as ovarian cysts [62]. Other ovarian malfunctions, such as ovarian haematoma, tumours and adhesions have been found in culled pigs [29,71,72]. However, there is no referred evidence in the literature that such conditions have ever been identified in situ via ultrasonography. We have published two cases of intersex pigs [73]. Both pigs were of female phenotype
and had multiple follicles on one ovary, while the other ovary was never detectable. The uteri contained small amounts of anechogenic fluid. Post-mortem examination revealed one intact ovary and a contralateral ovotestis, with uterine fluid accumulation as a result of congenital obliteration of one uterine horn. A third pig was later detected, had bilateral testicle-resembling structures within the abdominal cavity (Fig. 3F) and a malformed, rudimental uterus. Examination of uterine structures currently utilizes three criteria: fluid echogenicity, echotexture and size [31]. Any fluid echogenicity, unless attributed to pregnancy, semen or estrus [8], must be considered abnormal and indicative of an exudative inflammation of the uterus. Further delineation of this condition can range from endometritis [7], overt metritis [34] to pyometra [17]. In the pig, exudation and subsequent intrauterine fluid can be observed by ultrasonography if uterine inflammation is of an acute or acute-chronic type [30,31]. Although intrauterine fluid through those types of inflammation usually appears as ‘‘snow flakes’’ in the ultrasonographic image (Fig. 4A; [17,34]), an almost anechogenic appearance (Fig. 4B) or a tremendously heterogeneous echotexture of uterine cross-sections is also possible (Fig. 4C). In contrast, chronic endometritis, representing the most common type of uterine inflammation in pigs [30,31], cannot be definitively diagnosed by ultrasonography on the basis of any of the above-mentioned criteria [31]. To evaluate uterine echotexture it is absolutely necessary to consider physiological changes that occur during the estrous cycle and post-weaning. Accordingly, the uterine echotexture is heterogeneous at proestrus and estrus and more homogeneous at all other stages, including at post-weaning [11,33,34]. Changes in echotexture are a reflection of changes in the endometrial oedema [31], which is influenced by estrogens produced when developing follicles are present [33,72]. Consequently, in cases of females which have corpora lutea or only small follicles, endogenous estrogens are low and, thus, endometrial oedema is physiologically lacking [33,74]. Those relationships have been identically described in mares [75,76] and cows [49]. To evaluate the diagnostic value of uterine echotexture in the pig, repeat breeder pigs were fed Altrenogest for 15 days to hormonally mimic the diestrous phase of estrous cycle [32]. Females were than scanned at the end of treatment for uterine echotexture (graded 1–4 for homogeneous to strong heterogeneous) and the ovaries, and the ovulation process monitored to exclude ovulation failure. This study revealed that females having a strong heterogeneous uterine echotexture displayed reduced fertility
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Fig. 4. Images obtained by transcutaneous ultrasonography using a HS120 ultrasound machine (HONDA Electronics, Tokyo, Japan) and a 5.0 MHz linear-array transducer showing uterine cross-sections (arrows) from females (n = 3) having an exudative endometritis. In endometritis, intrauterine fluid can appear either flocculent (A) or anechoic (B). (C) An extremely heterogeneous uterine echotexture coupled with uterine enlargement. Images A and C were obtained from sows exhibiting a purulent vulvar discharge, and image B came from a female having a histologically diagnosed endometritis post-mortem. Scale bar on the top and left margin in 0.5 cm.
as determined by having the lowest pregnancy rate compared to females with other echotexture grades. In contrast, the uterine size, as expressed as the mean sectional area of uterine cross-sections [31], did not appear to correlate with pregnancy rate [32]. 4. Incorporation of ultrasonography of the female genital tract in pig production Given the great value of this diagnostic aid, it is inevitable that ultrasonography will be implemented into pig production beyond its current use. Monitoring of ovarian activity has frequently been suggested as an additional tool of ultrasonography in pigs. Examination to monitor ovulation, however, requires much experience, a higher amount of labour, and needs to be timed specifically for each individual female. Therefore, routine use of ultrasonography for monitoring ovulation seems therefore unlikely, with the procedure being performed on appointment, particularly in case whole herd insemination strategies need to be improved. This might be similarly true for determination of pubertal status in gilts. In contrast, pregnancy diagnosis by ultrasonography has already been successfully implemented into pig production and is routinely performed [8,17,34]. If pregnancy diagnosis is performed in early gestation, such as on day 20 or 21, there is the added advantage that any female not detected to be pregnant can be identified and increased estrus detection pressure placed on them [8,12,14,17,37]. Another advantage to pregnancy detection at 20–21 days is that pregnant females are identified and, thus, inappropriate estrus detection pressure and mating are avoided [14]. The major benefit, however, is if non-pregnant pigs would be
additionally examined for the ovaries and uteri. A great variety of ovarian structures has been observed in first served non-pregnant sows and gilts [42], which require individual approaches, such as immediate culling of females having polycystic ovarian degeneration or treatment of females having corpora lutea with PGF2a in an attempt to induce luteolysis and subsequent estrus [12]. Moreover, some uterine diseases, as indicated by abnormal intrauterine fluid or abnormal heterogeneous echotexture, can be diagnosed and these females immediately culled [31,32]. Although economic benefits need to be specified, such a procedure (i.e., the combination of pregnancy diagnosis and ovary as well as uterus examination) will certainly effectively contribute to reduce the number of non-productive sow days in a herd. Acknowledgements We are grateful to Juergen Eberspaecher and the staff of Physia GmbH (Neu-Isenburg, Germany) as well as Veyx Pharma (Schwarzenborn, Germany) for supporting many of Dr. Kauffold’s studies. We acknowledge the contribution of Dr. Tanja Rautenberg, Andreas Richter and Bent von dem Bussche to parts of the results presented in this review. References [1] Ginther OJ. Reproductive biology of the mare. Cross Plains: Equiservices; 1992. [2] Taverne MAM, Willemse AH. Diagnostic ultrasound and animal reproduction. Dodrecht, Boston, London: Kluwer Academic Publishers; 1989.
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[3] Inaba T, Nakazima Y, Matsui N, Imori T. Early pregnancy diagnosis in sows by ultrasonic linear-electronic scanning. Theriogenology 1983;20:97–101. [4] Botero O, Martinat-Botte´ F, Bariteau F. Use of ultrasound scanning in swine for detection of pregnancy and some pathological conditions. Theriogenology 1986;26:267–78. [5] Jackson GH. Pregnancy diagnosis in the sow using real-time ultrasonic scanning. Vet Rec 1986;119:90–1. [6] Fraunholz J, Ka¨hn W, Leidl W. Sonography for pregnancy diagnosis of swine—comparison between transrectal and transcutaneous procedures. Mh Vet Med 1989;44:425–30. [7] Martinez E, Vazquez JM, Roca J, Ruiz S. Use of real-time ultrasonic scanning for the detection of reproductive failure in pig herds. Anim Reprod Sci 1992;29:53–9. [8] Kauffold J, Richter A, Sobiraj A. Results and experiences of pregnancy control in swine by ultrasonography on different days of gestation over two years. Tiera¨rztl Prax 1997;25:429– 37. [9] Almond GW. Clinical reproduction in the sow: integrating new technology with stockmanship. In: Proceedings of the 15th IPVS Congress; 1998. p. 33–44. [10] Flowers WL, Armstrong JD, White SL, Woodhard TO, Almond GW. Real-time ultrasonography and pregnancy diagnosis in swine. Proc Am Soc Anim Sci 1999, http://www.asas.org/jas/ symposia/proceedings/0945.pdf. [11] de Rensis F, Bigliardi E, Parmigiani E, Peters AE. Early diagnosis of pregnancy in sows by ultrasound evaluation of embryo development and uterine echotexture. Vet Rec 2000;2:267–70. [12] Miller GM, Breen SM, Roth SL, Willenburg KL, Rodriguez-Zas A, Knox RV. Characterization of image and labor requirements for positive pregnancy diagnosis in swine using two methods of real-time ultrasound. J Swine Health Prod 2003;11:233–9. [13] Taverne MAM, Oving L, van Lieshout M, Willemse AH. Pregnancy diagnosis in pigs: a field study comparing lineararray real-time ultrasound scanning and amplitude depth analysis. Vet Quart 1985;7:271–6. [14] Kauffold J, Richter A, Rautenberg T, Sobiraj A. The ultrasonographic pregnancy diagnosis in pigs (1): a critical comparison of the procedures used to check for returns-to-service. Tiera¨rztl Prax 2003;31(G):336–41. [15] Szenci O, Taverne MAM, Palme R. Evaluation of ultrasonography and the determination of unconjugated oestrogen in faeces for the diagnosis of pregnancy in pigs. Vet Rec 1993;15:510–2. [16] Vos EA, van Oord R, Taverne MAM, Kruip TA. Pregnancy diagnosis in sows: direct ELISA for estrone in feces and its prospects for an on-farm test, in comparison to ultrasonography. Theriogenology 1999;51:829–40. [17] Knox RV, Althouse GC. Visualizing the reproductive tract of the female pig using real-time ultrasonography. J Swine Health Prod 1999;7:207–15. [18] Weitze KF, Habeck O, Willmen T, Rath D. Detection of ovulation in the sow using transcutaneous sonography. Zuchthygiene 1989;24:40–2. [19] Schmidt A. Studies on the control of ovarian cysts in sows using transcutaneous sonography. Dissertation. Hannover: School of Veterinary Medicine; 1993. [20] Gherpelli M, Tarocco C. A study on the incidence and clinical evaluation of the ovarian cysts in the sow. In: Proceedings of the 14th IPVS Congress; 1996. p. 587. [21] Castagna CD, Peixoto CH, Bortolozzo FP, Wentz I, Neto GB, Ruschel F. Ovarian cysts and their consequences on the
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Review
An update on the use of B-mode ultrasonography in female pig reproduction J. Kauffold a,*, G.C. Althouse b a
Large Animal Clinic for Theriogenology and Ambulatory Services, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 29, 04103 Leipzig, Germany b School of Veterinary Medicine, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348, USA Received 10 May 2006; received in revised form 23 October 2006; accepted 17 December 2006
Abstract After technological advances allowed for the adaptation of B-mode ultrasonography equipment for use in pig facilities, ultrasonography quickly established itself as an ideal diagnostic aid for determining pregnancy status in pigs. In recent years, Bmode ultrasonography has found increased application in its use for monitoring ovarian activity and in estimating time of ovulation in pigs. B-mode ultrasonography is also valuable in providing a detailed assessment of the sow’s ovaries and uterus to determine if pathological conditions exist, which could be contributing to poor individual or herd reproductive performance. In its most recent application in pigs, the gilt genital tract has been characterized peripubertally by ultrasonography in order to detect onset of puberty. The purpose of this review is to provide an update on the current status of B-mode ultrasonography in pig reproduction, and how this technology can be of value when used in pig production medicine. # 2007 Elsevier Inc. All rights reserved. Keywords: Pig; Ovary; Uterus; Puberty; B-mode ultrasonography
Contents 1. 2. 3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical requirements, use and equipment biosecurity . . . . . . . . . . . . . . . . . . . . Ultrasonographic examination of the female genital tract . . . . . . . . . . . . . . . . . . . 3.1. Ultrasonographic examination of the gravid female and pregnancy failure. . . 3.2. Ultrasonographic examination to monitor follicular dynamics and ovulation . 3.3. Ultrasonographic examination of the gilt and determination of puberty status 3.4. Ultrasonographic examination of the non-productive female . . . . . . . . . . . . Incorporation of ultrasonography of the female genital tract in pig production . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +49 3419738364; fax: +49 3419738398. E-mail address: [email protected] (J. Kauffold). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.12.005
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1. Introduction For many years ultrasonography has proven itself an excellent diagnostic tool for the gynaecological examination in many farm animal species [1,2]. In 1983, Inaba et al. [3] was one of the first who demonstrated the value of B-mode ultrasonography in diagnosing pregnancy in pigs. In the following years, multiple studies were performed [e.g., 4–12] assessing the use of B-mode ultrasonography in pigs. B-mode ultrasonography has repeatedly proven its superiority to other methods of pregnancy diagnosis such as service non-return rates [13,14] and endocrinological testing modalities [15,16]. Altogether, the pig industry has embraced this technology for diagnosing pregnancy in pig production operations [8,17], however, there still exist issues of dispute such as the day of first use postbreeding or the necessity for serial examinations on individual animals. In 1985, Botero et al. [4] reported the successful use of ultrasonography in pigs for the detection of ovarian cysts. It took a further 4 years until a research group demonstrated the use of transcutaneous B-mode ultrasonography in visualizing normal, peri-ovulatory ovarian structures and to monitor the ovulation process [18]. Further work corroborated the value of B-mode ultrasonography for diagnosing ovarian pathology [19– 21]. These applications have had a favourable impact both in their use in scientific studies [22–26], and much less so in pig production systems [9,17,27,28]. In contrast to its use in visualization of the ovary, only a few studies have applied B-mode ultrasonography in the assessment of the porcine uterus [4,7,11]. This is somewhat surprising given that uterine disorders account for a large percentage of reproductive failure in pigs [29,30]. Ultrasonography has been promoted in the assessment of intrauterine fluid, other than pregnancy, as an indicator of uterine inflammation and disease [7,17], and has shown value in providing detailed information on the overall health of the porcine uterus [31–33]. Eight years back Martinat-Botte´ et al. [34] presented a condensed text on ultrasonography in pig reproduction. Since then, there has been a large amount of new information generated on the topic. The purpose of this review is to summarize the current knowledge on the use of ultrasonography in assessing the reproductive tract in pigs. Along with reviewing its use in pregnancy diagnosis, special emphasis will be placed on new information garnered from recent investigations studying the use of B-mode ultrasonography in puberty [35,36] and on uterine pathology diagnosis [31,37].
Inferences to the value of this diagnostic aid in pig production and practice will be made. 2. Technical requirements, use and equipment biosecurity Several transducers have been used for gynaecological examination of the female pig, including lineararray [8,14,38] and convex-array transducers [17,39], as well as mechanical sectors [4,9,15]. Each transducer has particular advantages and disadvantages [10,17,40], with no one transducer clearly delineated as optimal for use in pigs. From the authors’ experience, most commercially available transducers have sufficient resolution and durability for use in pig practice, with personal preferences usually dictating which type is used. To date, there have been no reports in the scientific literature, which address specific requirements for ultrasound machines used in pig practice. According to the few available reports [17,40] and the authors’ experience, machines should be portable (i.e., lightweight and having an internal power supply) and robust. Portability is frequently regarded as a primary factor in machine choice given the mobility needed by personnel in using this equipment in group-housed or crated sows. For assessment of the sow reproductive tract, a transducer frequency of 5.0 MHz provides the most versatility [8,9,11,31–33]. Other frequencies, including 3.5 and 7.0 MHz, have been investigated for use in pregnancy diagnosis and ovary examination [3– 5,10,17]. Transabdominal/transcutaneous probe placement and scanning is commonly used in the industry for early, accurate diagnosis of pregnancy [6,11,38]. Transrectal ultrasonography has also been advocated for this purpose [40] and, in some aspects preferred [6,17]. In particular, some authors [17,22–25,28,41] favour transrectal scanning for assessing the ovaries. Comparison of transcutaneous and transrectal procedures was performed in the early 1990’s [41]. It was found that transcutaneous assessment was at a disadvantage for a number of reasons including difficulty in obtaining clear images due to interference with intestinal tissues, ambiguous detection and counting of corpora lutea, and the technical experience needed to perform the procedure properly. Moreover, repeated transrectal scanning has been found to not have an effect on reproductive outcomes such as fertilization and embryonic development [42]. Utilizing currently available equipment, the authors have successfully used transcutaneous scanning to detect puberty in gilts [36] and to characterize the ovaries [43] and uteri [31] in reproductively failed pigs. This route of scanning was
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found to be performed relatively simple, fast and safe procedure (e.g., due to no necessity to remove faeces [12] and of no risk of trauma to the animals [39,43]). Although ultrasound machines used in pig production operations have been identified as a biosecurity risk [16,17], only one recent study has addressed this issue [44]. According to this study [44], ultrasound equipment used in pig units was found to harbour several bacterial species, including the potentially pathogenic Streptococcus sp. Of higher interest, porcine reproductive and respiratory viral RNA was able to be recovered from the inside of different types of ultrasound machines. Given these findings, ultrasound equipment may carry a risk of being a fomite for the transmission of diseases. It is recommended that machines and all equipment be cleaned and disinfected after each use. While on the farm, equipment should be covered to aid in biosecurity, and transport of equipment between farms should be minimized. If travel between farms is necessary, sufficient sanitation protocols, including equipment downtime, should be used. 3. Ultrasonographic examination of the female genital tract 3.1. Ultrasonographic examination of the gravid female and pregnancy failure The earliest detection of pregnancy-specific structures (i.e., fluid-filled embryonic vesicles) has been reported to vary between 12 and 16 days of pregnancy [6,11,17,38]. Early pregnancy detection at 12–16 days can be, however, challenging in the sow using current transcutaneous ultrasonography techniques. As with other species, pregnancy detection rates in pigs improve as pregnancy progresses [8,11,17] and the conceptus increases in size [8,11]. While the developing conceptus has a 4.0 mm transverse diameter on day 15 of gestation, by days 18–22 the conceptus measures approximately 10 mm in diameter [11]. Using transrectal scanning and a 7.5 MHz probe, the embryo can usually be first detected on day 18 [12]. With transcutaneous scanning and a 5.0 MHz probe, pregnancy can be determined between 19 and 20 days of gestation [6,8]. Heartbeats appear between days 21 [6] and 25 [38], and are appropriate to determine viability of embryos or fetuses [9]. Transcutaneous Doppler cardiography has been used to study fetal heart rate (FHR) of eight fetuses in the last 10 days of gestation [45]. FHR decreased in seven normal fetuses toward parturition, but increased in a fetus with abdominal ascites [45] suggesting that antepartum Doppler FHR
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measurement might be feasible to predict viability of fetal and newborn pigs [46]. Both the fetal eye orbit and the stomach have been first detected on day 49 of gestation by transcutaneous scanning using a frequency of 5.0 MHz [6]. The diameter of the fetal heart, eye orbits and stomach grow at a linear rate (r = 0.75–0.92), and estimation of their transverse or longitudinal dimension can be helpful in determining gestational stage if breeding dates are unknown [6]. In pregnant cattle, endometrial echogenicity (expressed as the mean grey level) declined between days 7 and 13 after ovulation, followed by a subsequent moderate rise reaching medium values on day 15 [47]. These same changes in endometrial echogenicity were not present in cyclic cows [48,49]. Similarly, a decline in echogenicity of the cross-sectional image of the uterine horn was observed in pregnant gilts between days 12 and 14 of gestation, but was absent in nongravid, control gilts (Fig. 1; J. Kauffold, B. von dem Bussche, A. Sobiraj, M. Wendt; unpublished observation). This decline in echogenicity might be due, at least in part, to an increased endometrial oedema, as only uterine cross-sections being completely devoid of luminal fluid were measured. Preliminary work by the authors suggests that this temporal change in endometrial echogenicity might have value in the indirect diagnosis of early pregnancy and embryonic mortality. However, the level of training and necessary equipment resolution may preclude its use as a routine means for very early pregnancy diagnosis in pigs.
Fig. 1. Echogenicity of uterine horn cross-sections expressed as the mean grey level S.D. in pregnant (squares; n = 4) and non-pregnant (circles; n = 8) gilts on days 5–16 post-ovulation (day 0). Echogenicity was estimated using grey scale analysis (grey levels ranged from 0 to 32) with a HS 2000 (HONDA Electronics, Tokyo, Japan), a 5.0 MHz linear-array transducer and transcutaneous scanning. For analysis, a mean of 6.4 entire uterine cross-sections per gilt were estimated using standardized equipment settings. Pregnant females had a significantly (P < 0.05) lower echogenicity between days 12 and 14 than the nonpregnant females (values with letters (a and b) differ; J. Kauffold, B. von dem Bussche, A. Sobiraj, M. Wendt; unpublished observation).
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Successful diagnosis of pregnancy status in early gestation depends upon transducer frequency, route of application, technical expertise, experience and other factors inherent to the patient themselves [9,17]. For instance, correct diagnosis of pregnancy status was higher on day 21 if a 5.0 MHz transducer was used transcutaneously versus a 3.5 MHz transducer [9,10]. A transrectal approach allowed earlier pregnancy diagnosis with greater accuracy than examinations done by the transcutaneous procedure [6]. In general, recognition of pregnancy is easier than non-pregnancy [8,10]. While pregnancy-specific structures definitely indicate pregnancy, their absence does not necessarily indicate a non-gravid status, as in some pregnant females, particularly during early stages, structures indicative of pregnancy can remain undetected [6]. Pregnancy diagnosis can approach 100% sensitivity as early as day(s) 19 [11], 20–21 [8] or 21–23 [9] of gestation. Specificity (i.e., ability to distinguish from a non-gravid sow), however, was almost always significantly lower, irrespective of the stage of gestation [6–9]. The main reason for the low specificity is that females initially identified as pregnant by ultrasonography and later returned to estrus or failed to farrow were presumed to be false positives [8,10,11,37]. This presumption, however, can be erroneous as those females identified as pregnant at the time of initial examination may have indeed lost pregnancy thereafter [8,10–12,37]. Thus, ultrasonography is maybe much more accurate in the
diagnosis of non-pregnant animals than the available data suggests [8,37]. The necessity of re-confirming pregnancy at a later date in females initially diagnosed early in gestation is a current area of debate [7,8,11,37]. This is in lieu of the fact that the rate of embryonic mortality in sows is highest up until day 30 of gestation [50,51]. Recent work, however, suggests that re-confirming pregnancy on a routine basis may be of limited value given that the number of females diagnosed pregnant and later farrowed were found to be non-significantly different [37]. Ultrasonography has clinical value for diagnosing pregnancy failure and early embryonic death leading to either fetal mummification [12] or partial embryonic decomposition (Fig. 2A; [8]). Ultrasonography can be valuable in assessing females which had recently aborted or had undergone complete embryonic loss based upon the echogenicity and non-typical shape of the fluid-filled uterine structures devoid of the embryo proper or enlarged uteri exhibiting extreme heterogeneous echotexture [37]. Diagnosis is aided from additional information such as data on breeding and/or clinical signs observed. Another, but rather uncommon diagnosis is hydrometra, which has been observed twice among approximately 3 105animals scanned by us for pregnancy during the last 12 years. In both cases of hydrometra, the uterus was enlarged with thinning of the uterine wall through extreme intrauterine accumulation of fluid (Fig. 2B).
Fig. 2. Images obtained by transcutaneous ultrasonography using a HS120 ultrasound machine (HONDA Electronics, Tokyo, Japan) and a 5.0 MHz linear-array transducer. (A) Decomposing embryo (arrow; taken from [8]). Note the loss in homogeneous echotexture and mottled appearance. (B) Hydrometra in a sow. The uterus is filled with large amounts of fluid, which has lead to an extreme thinning of the uterine wall (walls of two adjacent uterine horn cross-sections are delineated between arrows). Scale bar on the top and left margin in 0.5 cm.
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3.2. Ultrasonographic examination to monitor follicular dynamics and ovulation From recent reviews [52–55], ultrasonography has greatly increased our understanding of follicular dynamics in pigs. Most research has been done in the lactating pre-weaned and in the post-weaned sow. According to Lucy et al. [52], pre- and post-weaning follicular growth displays a wave-like pattern. This wave consists of a cohort of 20–30 follicles of 2 mm in size that synchronously starts to grow in response to FSH stimulation. During lactation, co-dominant follicles reach up to 5 mm and then regress. Post-weaning, those follicles appear to contribute to the pre-ovulatory pool [52]. Developmental stage of follicles at weaning appears to influence weaning-to-estrus interval (WEI), as females having follicles being in the developmental stage of a cohort come into estrus earlier than females having follicles of similar size but weaned when the follicular cohort was regressing [50]. The size of the follicles at weaning seems to be equally important, as a reduced follicle size on day 2 or 3 after weaning was associated with an increased WEI and weaning-toovulation interval [24,56]. Support for those observations also comes from a recent study [57] showing that sows treated pre-weaning with multiple injections of charcoal-extracted porcine follicular fluid, aimed to suppress FSH and follicular growth, had a mean follicle size at weaning smaller than that of saline-treated controls (1 versus 3 mm) and a longer WEI (6.1 0.4 versus 4.7 0.4). The relationship, however, does not appear to be strong enough to be used to predict estrus or ovulation on the basis of follicle size determined around weaning [24,56]. Through ultrasonography a number of factors have been identified in association with reduced follicle size peri-weaning, including high ambient temperature [52] and low energy in feed [58] during lactation, first parity and bad body condition [24]. Once the post-weaning follicular growth has emerged, the dynamics of follicular development (i.e., the slope) appear to be unaffected by follicular size around weaning [24,52]. At the beginning of estrus [23,28] and 20 h thereafter [56], pre-ovulatory follicles average between 7 and 9 mm [56], but size can vary considerably between females. Soede et al. [56] observed that follicles measuring <7 mm 16 h prior to ovulation increased in size up to ovulation, while follicles >7 mm do not change or become somewhat smaller. Pre-ovulatory follicle size does not appear to affect reproductive traits, as no association was found between follicle size and WEI, fertilization rate, as well as embryo development and diversity [56]. Minor
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reduction in size and changes from a spherical to a more ovoid [52,56] or longish shape [57] of the pre-ovulatory follicles immediately preceding ovulation have been reported, with a suggested loss in follicle turgidity [56,59], similar to that observed in mares [60]. These changes have been proposed to be candidate parameters to predict ovulation time in pigs [59]. However, there appears to be no reports in the scientific literature to support this. Considering the importance follicular turgidity plays in determining ovulation time in mares [60], it might be worthy of further investigation in pigs. Initiation of ovulation and the inter-ovulatory period has been estimated using sequential examinations of the ovaries to follow a reduction in the number of follicles [17,28]. The ovulation process can take up to several hours [22,61], and pre-ovulatory follicles can be observed concomitant with corpora haemorrhagica [43]; the latter are clearly identified by characteristic echogenic appearance [43]. Ovulation was considered to be completed if sequential examinations showed the disappearance of all pre-ovulatory follicles [17,21,39] or a marked reduction in the number of pre-ovulatory follicles relative to previous observations [28], or the ovary could not be visualized [62]. Failure to visualize the ovary, however, seems to be a rather unreliable criterion given that in some situations the ovaries can not be located due to circumstances not related to disappearance of follicles [43]. The presence of corpora haemorrhagica while pre-ovulatory follicles are absent seems to be most indicative for ovulation completion [31,36,43]. Different intervals between examinations, ranging from 4 to 24 h, have been chosen for monitoring ovulation in pigs [28,56]. Although the closer the intervals are logically more precise in the diagnosis of ovulation time [28], the longer interval of 24 h is more desirable from a practical standpoint, and seems to be adequate when used appropriately [23,28]. A number of authors have emphasized that implementation of ultrasonography for monitoring ovulation in pig production would be economically beneficial (e.g., proper timing of ovulation, insemination timing, avoiding post-ovulatory inseminations, etc. [9,17,27,40,62]). Although the inherent difficulty in routine application of this technique in the field has precluded its widespread implementation so far, examples in the literature suggest that its application is increasing in practice [27,28,40]. The greatest value of monitoring ovulation in a farm setting is certainly to improve insemination strategy [9,28,40,62]. Almond [9] speculated that producers might be able to perform ultrasonography to monitor ovulation and might then inseminate sows individually.
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Fig. 3. Images obtained by transcutaneous ultrasonography using a HS120 ultrasound machine (HONDA Electronics, Tokyo, Japan) and a 5.0 MHz linear-array transducer. (A) Polycystic ovarian degeneration (POD): multiple, approximately 2.5 cm measuring anechogenic spherical structures without accompanying corpora lutea (CL). (B) POD, with cysts having a 3 mm thick wall (delineated between arrows) suggestive of luteinized tissue. (C) A 2.5 cm large ‘‘blood cyst’’ having similar echogenicity as a CL (refer to image D), but with an irregularly shaped small anechogenic cavum. Insert shows the same ovary post-mortem. (D) Oligocystic ovarian degeneration: two cysts (black arrows) accompanied by two clearly
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3.3. Ultrasonographic examination of the gilt and determination of puberty status Ultrasonography has been proven an appropriate tool for the diagnosis of puberty status in gilts. Both the evaluation of the ovaries and the uterus have been found suitable in determining gilts as either prepubertal or pubertal. Follicles of 2–5 mm in size and absence of any luteal structures in the ovary is a typical finding for prepubertal females [17,36]. Uterine size may also be of value in determining gilt sexual maturity. MartinatBotte´ et al. [35] collected uterine horn vertical diameters and employed an integrated computer program to calculate the ‘‘uterine area’’, i.e., an area covering all visible parts of the uterus within an ultrasound image. They estimated a sensitivity and specificity of 98.8 and 100%, respectively, for the diagnosis of prepubertal and pubertal gilt status. Kauffold et al. [36] also utilized uterine size in determining pubertal status, but measuring two or more cross-sections of the uterine horns in their maximum and minimum dimensions. Cross-sectional areas were calculated using the mathematical equation for an elliptical figure, and averaged all areas to give the mean sectional area of the uterine horns for a female. Accordingly, prepubertal females were found to have a cross-sectional area of 1 cm2, with pubertal animals exhibiting cross-sectional areas of 1.2 cm2. These determination values were then applied in a field study and results verified on the basis of progesterone blood concentrations and estrous behaviour. Accuracy of pubertal and prepubertal diagnosis was 91.0 and 100%, respectively. 3.4. Ultrasonographic examination of the nonproductive female During recent years, progress has been made in the use of ultrasonography to examine the genital tract of non-productive sows in order to detect reproductive disorders [17,34] and, more specifically, ovarian disorders and the diseased non-gravid uterus. Recognition of ovarian disorders in pigs is possible through the fact that physiological ovarian structures are frequently well-defined by ultrasonography [17,42,63]. Diagnosis of cystic ovarian disease is relatively simple [17,19,27,43], but complete delineation of ovarian cyst type via the ultrasonographic image can vary somewhat.
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Ovarian cysts are defined as fluid-filled structures that exceed a diameter of 11 [27] or 12 mm [28,64], as unovulated or luteinized follicles of 12–50 mm appearing large, smooth, circular and anechogenic [17], or as anechogenic structures with smooth, thin walls of 20 mm in diameter being present at least for 5 days after ovulation [21]. Follicular cysts, originating from unovulated follicles, usually appear as fluid-filled bubbles surrounded by a thin wall (Fig. 3A; [17,19,27,43,59]). In contrast, luteal cysts, originating from a developed corpora lutea, have a rather thick wall and a distinct lumen (Fig. 3B), which sometimes contains trabeculae-like structures [20], and are thus rather dissimilar to so called ‘‘blood cysts’’ (Fig. 3C) assumed to develop from overgrown corpora haemorrhagica [27,65]. The origin of luteinized cysts, if diagnosed, is impossible to determine since follicular cysts are also known to luteinize spontaneously [66,67]. Information on the cyst origin, however, is of lesser interest than is the observation of a luteinized wall per se, as it might indicate an ongoing regression process [21,43,67] and/or susceptibility of exogenous PGF2a administration. Besides cyst type, ovarian cysts can be distinguished on the basis of their number, as both single [19,43] and multiple cysts (i.e., 2, also referred as to oligocystic ovarian degeneration when accompanied by corpora lutea; Fig. 3D; [19,21,43]) have been described. Using ultrasonography, those conditions were estimated to occur between 7.6% [43] and approximately 30% [27] in breeding pigs and occasionally also observed preweaning [52], and consequences on reproductive performance such as reduction in litter size [27], lower farrowing rate [21,27] and higher frequency of returns to estrus [21] have been reported. Polycystic ovarian degeneration [17,19,21,43], also referred as to cystic follicular degeneration [27], is characterized by multiple fluid-filled bubbles (i.e., cysts) and the absence of corpora lutea [43]. This condition has been estimated to occur between 0.6 [27] and 4.3% [43] in breeding pigs on the basis of ultrasonographic examinations. It normally blocks reproductive function life-long and is a valid reason to cull females immediately. Individual sow cases of spontaneous regression, albeit after several weeks, have however been reported [66]. Another ovarian malfunction is ovulation failure. Pre-weaning ovulation was observed by Lucy et al. [52] in a small percentage of sows and certainly affects
recognizable CL (white arrows). (E) A para-ovarian cyst (arrow) of irregular shape and approximately 2 cm in diameter attached to an ovary having several large, pre-ovulatory follicles. (F) Intra-abdominal testicle-resembling structure found in a gilt. The structure is completely homogeneous in echogenicity, typical of intact testicles in the male. Insert shows the structure post-mortem. Scale bar on the top and left margin in 0.5 cm.
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expression of fertile estrus post-weaning. Delayed ovulation, defined as a follicle(s) that ovulate(s) 7 h after the majority of follicles has ovulated [19,27], has been observed in 3.8% out of 346 females using repeated ultrasonographic examination [19]. This condition, however, does not appear to affect reproductive performance [19,27]. Persistence of single medium sized follicles distinct from ovarian cysts beyond ovulation has been reported to occur occasionally in sows [22]. Complete ovulation failure has been diagnosed in weaned sows detected in standing estrus [23], and was associated with a short lactation length of 16 days [28]. Follicular size was inconsistent, as those sows could have variable sized follicles ranging from small to medium [28] or of pre-ovulatory size [23]. Whether inactive ovaries and corpora lutea (pseudo)persistent (CLP) are primarily ovarian malfunctions is a matter of question. Inactive ovaries, characterized by several follicles of predominantly smaller size, were one major finding in non-pregnant first service sows and gilts scanned between days 20 and 114 after breeding [43], and those females usually reported being anestrous for a longer interval [32,43]. Failed follicle growth has been repeatedly observed by ultrasonography in sows after weaning. Insufficient gonadotropin support (i.e., LH), necessary for final follicular maturation, was suggested as the main reason for failure [23,28,52]. Among those sows experiencing failed follicular growth, two distinct populations have been described, one without any post-weaning follicle growth, and the other with follicle growth up to 4 or 5 mm without further progression to a pre-ovulatory size [23,28,52,68]. CLP might be the result of embryonic mortality [23] or exposure to zearalenone [69,70] and do not differ from cyclic corpora lutea in the ultasonographic appearance [23,32]. Differentiation might only be possible if additional information (such as on last estrus/breeding) is available and pregnancy definitely excluded. Para-ovarian cysts have been detected by ultrasonography in 18 of 346 females scanned [19,28]. They are of different size (>1 cm up to several cm), completely anechogenic, but variable in shape (round, ovoid, polygonal; Fig. 3E). They do not appear to impair reproduction function and can easily be source for misdiagnosis as ovarian cysts [62]. Other ovarian malfunctions, such as ovarian haematoma, tumours and adhesions have been found in culled pigs [29,71,72]. However, there is no referred evidence in the literature that such conditions have ever been identified in situ via ultrasonography. We have published two cases of intersex pigs [73]. Both pigs were of female phenotype
and had multiple follicles on one ovary, while the other ovary was never detectable. The uteri contained small amounts of anechogenic fluid. Post-mortem examination revealed one intact ovary and a contralateral ovotestis, with uterine fluid accumulation as a result of congenital obliteration of one uterine horn. A third pig was later detected, had bilateral testicle-resembling structures within the abdominal cavity (Fig. 3F) and a malformed, rudimental uterus. Examination of uterine structures currently utilizes three criteria: fluid echogenicity, echotexture and size [31]. Any fluid echogenicity, unless attributed to pregnancy, semen or estrus [8], must be considered abnormal and indicative of an exudative inflammation of the uterus. Further delineation of this condition can range from endometritis [7], overt metritis [34] to pyometra [17]. In the pig, exudation and subsequent intrauterine fluid can be observed by ultrasonography if uterine inflammation is of an acute or acute-chronic type [30,31]. Although intrauterine fluid through those types of inflammation usually appears as ‘‘snow flakes’’ in the ultrasonographic image (Fig. 4A; [17,34]), an almost anechogenic appearance (Fig. 4B) or a tremendously heterogeneous echotexture of uterine cross-sections is also possible (Fig. 4C). In contrast, chronic endometritis, representing the most common type of uterine inflammation in pigs [30,31], cannot be definitively diagnosed by ultrasonography on the basis of any of the above-mentioned criteria [31]. To evaluate uterine echotexture it is absolutely necessary to consider physiological changes that occur during the estrous cycle and post-weaning. Accordingly, the uterine echotexture is heterogeneous at proestrus and estrus and more homogeneous at all other stages, including at post-weaning [11,33,34]. Changes in echotexture are a reflection of changes in the endometrial oedema [31], which is influenced by estrogens produced when developing follicles are present [33,72]. Consequently, in cases of females which have corpora lutea or only small follicles, endogenous estrogens are low and, thus, endometrial oedema is physiologically lacking [33,74]. Those relationships have been identically described in mares [75,76] and cows [49]. To evaluate the diagnostic value of uterine echotexture in the pig, repeat breeder pigs were fed Altrenogest for 15 days to hormonally mimic the diestrous phase of estrous cycle [32]. Females were than scanned at the end of treatment for uterine echotexture (graded 1–4 for homogeneous to strong heterogeneous) and the ovaries, and the ovulation process monitored to exclude ovulation failure. This study revealed that females having a strong heterogeneous uterine echotexture displayed reduced fertility
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Fig. 4. Images obtained by transcutaneous ultrasonography using a HS120 ultrasound machine (HONDA Electronics, Tokyo, Japan) and a 5.0 MHz linear-array transducer showing uterine cross-sections (arrows) from females (n = 3) having an exudative endometritis. In endometritis, intrauterine fluid can appear either flocculent (A) or anechoic (B). (C) An extremely heterogeneous uterine echotexture coupled with uterine enlargement. Images A and C were obtained from sows exhibiting a purulent vulvar discharge, and image B came from a female having a histologically diagnosed endometritis post-mortem. Scale bar on the top and left margin in 0.5 cm.
as determined by having the lowest pregnancy rate compared to females with other echotexture grades. In contrast, the uterine size, as expressed as the mean sectional area of uterine cross-sections [31], did not appear to correlate with pregnancy rate [32]. 4. Incorporation of ultrasonography of the female genital tract in pig production Given the great value of this diagnostic aid, it is inevitable that ultrasonography will be implemented into pig production beyond its current use. Monitoring of ovarian activity has frequently been suggested as an additional tool of ultrasonography in pigs. Examination to monitor ovulation, however, requires much experience, a higher amount of labour, and needs to be timed specifically for each individual female. Therefore, routine use of ultrasonography for monitoring ovulation seems therefore unlikely, with the procedure being performed on appointment, particularly in case whole herd insemination strategies need to be improved. This might be similarly true for determination of pubertal status in gilts. In contrast, pregnancy diagnosis by ultrasonography has already been successfully implemented into pig production and is routinely performed [8,17,34]. If pregnancy diagnosis is performed in early gestation, such as on day 20 or 21, there is the added advantage that any female not detected to be pregnant can be identified and increased estrus detection pressure placed on them [8,12,14,17,37]. Another advantage to pregnancy detection at 20–21 days is that pregnant females are identified and, thus, inappropriate estrus detection pressure and mating are avoided [14]. The major benefit, however, is if non-pregnant pigs would be
additionally examined for the ovaries and uteri. A great variety of ovarian structures has been observed in first served non-pregnant sows and gilts [42], which require individual approaches, such as immediate culling of females having polycystic ovarian degeneration or treatment of females having corpora lutea with PGF2a in an attempt to induce luteolysis and subsequent estrus [12]. Moreover, some uterine diseases, as indicated by abnormal intrauterine fluid or abnormal heterogeneous echotexture, can be diagnosed and these females immediately culled [31,32]. Although economic benefits need to be specified, such a procedure (i.e., the combination of pregnancy diagnosis and ovary as well as uterus examination) will certainly effectively contribute to reduce the number of non-productive sow days in a herd. Acknowledgements We are grateful to Juergen Eberspaecher and the staff of Physia GmbH (Neu-Isenburg, Germany) as well as Veyx Pharma (Schwarzenborn, Germany) for supporting many of Dr. Kauffold’s studies. We acknowledge the contribution of Dr. Tanja Rautenberg, Andreas Richter and Bent von dem Bussche to parts of the results presented in this review. References [1] Ginther OJ. Reproductive biology of the mare. Cross Plains: Equiservices; 1992. [2] Taverne MAM, Willemse AH. Diagnostic ultrasound and animal reproduction. Dodrecht, Boston, London: Kluwer Academic Publishers; 1989.
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