Evaluation of ovarian blood flow by colour Doppler ultrasound: practical use for reproductive management in the cow

Transrectal real-time ultrasonography (US) has been developed as a research and practical tool in bovine reproduction. Non-invasive US observations have made it possible to provide real-time and serial analyses of ovarian morphological changes and fetal development and have generated new information on reproductive physiology during the bovine oestrous cycle and pregnancy. This has greatly contributed to an understanding of the real-time dynamics of follicular development. US has also allowed for more accurate diagnosis compared with rectal palpation in reproductive management in cattle. Practical applications of US include early diagnosis of pregnancy, identification of twin fetuses, detection of ovarian and uterine pathologies and determination of fetal sex.In recent years, local blood flow has been analysed in individual ovarian follicles and the corpus luteum (CL) in the cow using colour Doppler US. From these observations, it has been found that (1) the blood supply to follicles is closely related to follicular growth, atresia and ovulation, (2) the blood supply to the CL increases in parallel with its growth, and (3) there is an acute increase in blood flow in the mature CL prior to luteal regression. Colour Doppler US may provide an estimate of the physiological status of follicles and corpora lutea. For example, images of blood flow can be used to assess the thickness of the follicular wall and provide a differential diagnosis of follicular and luteal cysts. Assessment of the area of blood flow in the CL using colour Doppler imaging may offer a useful adjunct in estimating CL function, which could be applied to the diagnosis of non-pregnancy and fetal loss. The number of small follicles which have blood flow at the start of gonadotrophin treatment may be a useful index to predict the superovulatory response.With improvements in portability and cost-effectiveness, the evaluation of ovarian blood flow by colour Doppler US is likely to become widely used as a diagnostic tool for monitoring ovarian function in dairy cattle.     

超声波影像诊断技术在牛繁殖中的应用

本文详细阐述了超声波影像诊断技术,在卵泡测、妊娠诊断、胎儿性别鉴定、卵泡穿刺、生殖器官诊断、胎膜穿刺、雄性生殖力检查等牛繁殖领域的应用现状,并论述了存在的问题与不足。     

A potential use of color ultrasound as a tool for reproductive management: New observations using color ultrasound scanning that were not possible with imaging only in black and white

Ultrasonography (US) has been applied to the ovary and the uterus of domestic animals from the late 1980s, and established in 1990s as a practical tool for animal production. US made it possible to detect pregnancy at a very early stage and, most importantly, to observe the real-time dynamics of follicular development and hence the discovery of follicular waves. This has greatly contributed to our understanding of ovarian physiology and helped us to develop several "pin-point" protocols for hormonal treatment. While US may not seem to fit preconceived ideas of a "green" technology, it does not contravene environmental priorities, and it is non-invasive ("ethical") and non-hormonal ("clean"). Using the US technology that is now commercially available at a reasonable price, we are able to estimate the best timing for AI and this allows us to plan either the use of precisely-timed nutritional supplements for fetal development or an immediate 2nd AI service to achieve a better economic efficiency. During the last few years, we have also begun to be able to observe in detail the local blood flow in individual ovarian follicles and CL using color Doppler ultrasonography in the cow. From the series of observations, we have found that: 1) the change of blood supply to an individual follicle closely relates to the dynamics of follicular growth and atresia; 2) the local blood flow detected in the theca externa of mature follicles rapidly increases around the onset of LH surge and is most active before ovulation; 3) the blood supply to the developing CL increases in parallel with CL volume and plasma progesterone concentrations; and 4) the local blood flow surrounding the mature CL acutely increases prior to the onset of luteolysis in response to uterine as well as exogenous PGF(2alpha). It is now clear that color Doppler ultrasound is very useful for observing echogenicity with local blood flow thereby providing an easily obtained estimation of the physiological status of follicles, CLs and early conceptus. Widespread commercial application of color US will depend on further technological developments that reduce the cost and improve performance and ease-of-use. Overall, US is now a most effective non-invasive tool for managing reproduction, at the level of both the individual animal and the herd system. In particular, US can help us to clarify potential problems in high-producing dairy cattle during the postpartum period.     

Light microscopical and ultrastructural characterization of black howler monkey (Alouatta caraya) ovarian follicles

The present study describes the morphological characteristics of black howler monkey (Alouatta caraya) ovarian follicles. One ovary of an adult healthy black howler monkey was collected and processed for light and electron microscopy. Primordial, primary, secondary, tertiary and pre-ovulatory follicles were evaluated for their morphometrical aspects. The ovary of black howler monkey presented a distinct conformation with a uniform distribution of the follicles mostly in the peripheric cortex. This black howler monkey ovary presented a total of 59 921 ovarian follicles. From this amount, 71.1% were classified as primordial, 18.9% as primary, 8.1% as secondary, 1.4% as tertiary and 0.5% as pre-ovulatory follicles. From all these developmental stages, the mean diameters of follicles, oocytes, oocytes nuclei and the mean number of granulosa cells are described. Moreover, primordial, primary and secondary follicles have been observed by electron microscopy.     

休情期银黑狐卵巢形态和卵泡发育的显微结构观察

研究休情期银黑狐卵巢形态和卵泡的显微结构,以揭示银黑狐卵巢发育的一般规律。本试验于2012年12月份采集5只健康一岁龄银黑狐卵巢10枚,用游标卡尺测量其长、宽、厚,用电子天平测量其重量,并对其表面可见卵泡数量进行统计,然后利用光学显微镜对各级卵泡分别观察1~3个,共计原始卵泡30个,初级卵泡20个,次级卵泡15个,三级卵泡12个,成熟卵泡10个,并进行拍照。结果表明:随银黑狐卵巢体积不断增大,其中80%的卵巢重量也随之增大;可见卵泡数量与卵巢体积及重量没有相关性;卵巢由被膜、皮质和髓质构成,髓质位于卵巢内层,分布着较多血管,皮质位于卵巢外层,内有不同发育阶段的卵泡;原始卵泡由卵母细胞和颗粒细胞构成,初级卵泡开始出现透明带物质,到次级卵泡阶段发育完整,三级卵泡出现卵泡腔,卵泡及卵母细胞直径在有腔卵泡阶段比腔前卵泡阶段增长速度快,成熟卵泡的直径及透明带厚度达到最大,各级卵泡均有闭锁现象。     

Application of ultrasonography in the study of the reproductive system of tropical jennies (shape Equus asinus)

The use of high-frequency (5 MHz) ultrasonography was studied in 11 jennies (7 non-pregnant and 4 pregnant) to characterize the reproductive organs and follicular activities at different stages of reproduction. The result showed close similarity with the mare. The visibility of endometrial folds increased towards ovulation. A positive correlation (p < 0.001; τ = 0.79) was found between the score of the folds and the size of the dominant follicle. The diameter of the uterus and the size of the dominant follicle were significantly correlated (p < 0.001; τ = 0.80). In pregnant jennies, an embryonic vesicle was detectable at 14 days. Follicular growth was characterized by more than one wave. The smallest ovarian follicle was 2 mm and the largest 40 mm. Depending on the reproductive stage, up to 13 follicles were detected per ovary. After monitoring 84 cycles, a mean (±SD) diameter of 34.4 ± 3.6 mm (27.5–40.2 mm) of the preovulatory follicle and 67.85% incidence of single ovulation were found. The mean (±SD) interovulatory interval was 25.7 ± 6 days. This study proved that high-frequency ultrasonography is highly effective in characterizing the reproductive organs and follicular activity of jennies and could be useful in the reproductive management of donkeys.     

The pattern of ovarian development in the prepubertal antarctic minke whale (Balaenoptera bonaerensis)

This study describes the morphological and morphometrical changes associated with prepubertal ovarian development in the Antarctic minke whale (Balaenoptera bonaerensis). Ovaries were harvested from 94 immature minke whales caught in the Antarctic Ocean during the summer feeding season (December-March). Notable differences in ovarian size and morphology were found among animals. Up to 10 folds difference in ovarian weight was found among prepubertal whales of similar body size. During the prepubertal period, ovaries grew slowly and approximately doubled their weight. The morphologies of right and left ovaries were almost identical while the growth of the ovary appears to occur preferentially on the right side. The most striking morphological feature was numerous small antral follicles less than 5 mm in diameter found in ovaries of younger immature whales. The occurrence of these ovaries was highest in whales less than 6 m long and gradually decreased as body length increased. In larger whales, the occurrence of ovaries with a smaller number of follicles up to 10 mm and thick tunica albuginea increased. Thus, the ovary of the Antarctic minke whale experiences bursts of small follicular development during the early prepubertal period before becoming a more developed ovary with fewer but larger follicles, and thick tunica albuginea.     

A huge ovarian cyst in a hysterectomized bitch

A 11-year-old, spayed, female mixed breed-dog was presented with an abdominal mass that was detected 1 month ago. Upon abdominal palpation a large, firm, oval shaped, movable mass was found in the mid-abdominal region. Survey radiograph of the abdomen demonstrated an oval soft tissue dense mass located on the right side of the abdominal cavity. A large, heteregenous and cystic mass with solid components occupying the majority of the abdomen and a small, cystic mass with solid components caudal to the left kidney were identified by transabdominal ultrasonography. Computed tomography scans revealed bilateral ovarian masses, and a small volume of retroperitoneal fluid on the right side. A cystic, but otherwise solid mass located in the right ovary and small retained left ovary encapsulated in the ovarian bursa were excised surgically by midline laparotomy. Histopathological examination of the excised mass from the right side revealed a large cystic structure consistent with an ovarian cyst and multiple corpora lutea and follicles at different maturational stages were detected in the left ovary. The precise origin of the ovarian cyst could not be determined by morphological appearance. Immunohistochemical staining suggested a cyst of surface epithelial origin. At re-examination 6 months after the surgery, the bitch appeared healthy and the clinical findings were all normal. To our knowledge, the cyst described here is the largest reported in an incompletely ovariohysterectomized bitch.     

Relationships among the corpus luteum,follicles and conceptus in sheep

This study aimed to investigate the intra- and interovarian relationships among the corpus luteum (CL), the largest follicle (LF) and follicular population in non-pregnant and between the conceptus and ovarian structures in pregnant ewes. In experiment 1, the follicular and luteal structures were examined in 538 reproductive systems of non-pregnant Awassi ewes. The follicular population was categorised into small (SF), medium (MF) and large (LF) groups. Inter-relationships between CL and follicular population and between LF and subordinate follicles were determined. In experiment 2, the location and number of conceptuses were identified and correlated with the ovarian structures in 58 reproductive systems of pregnant ewes. Effects of pregnancy status, stage of pregnancy, pregnancy side and conceptual number on follicular population were determined. The results showed that the right ovary was more active than the left ovary. CL had intraovarian positive effect on the number of medium and large follicles. LF had no local suppressive effect on the subordinate follicles. Side and stage of pregnancy and the conceptual number did not affect the follicular population. Accordingly, it can be concluded that the LF has no local suppressive effect on the subordinate follicles. The CL has intraovarian positive effect on the follicular population. Follicular population does not show remarkable changes during the first term of pregnancy. The present study probably provides information which may help in the understanding of the ovarian dynamics during pregnancy in sheep.     

Ultrasonographic findings of an ovarian abscess in a cow

Ultrasonographic examination was carried out in a cow with an ovarian abscess. Real time ultrasound scanning using a 7.5 MHz linear probe per rectum revealed an active left ovary with follicles of up to 11 mm in diameter and an enlarged right ovary measuring 6 x 6 cm, with a mass of 4 x 3 cm. The mass had varied echotexture, a thick hyperechoic boundary and a hypoechoic centre with echoic foci. It was tentatively diagnosed as an ovarian tumour or abscess. After ovariectomy, grossly the right ovary measured 6 x 6 x 5 cm and on cutting cheesy pus oozed out. The abscess was completely localised within the ovary, 4 cm in diameter and encapsulated thickly. On bacteriological examination Staphylococcus spp. was isolated. This is a rare case of ovarian abscess.     

不同周龄蛋鸡卵巢机能及氧化还原状态的变化研究

本试验旨在研究不同周龄蛋鸡卵巢机能及氧化还原状态的变化规律。选择17周龄京红蛋鸡48只、78周龄京红蛋鸡24只,2只1笼为1个重复,分别饲养至36、48、97周龄采样。结果发现,随着周龄增大,蛋鸡卵巢机能衰退,表现为卵巢内等级卵泡、小黄卵泡、初级卵泡和次级卵泡数量显著减少(P<0.05),小黄卵泡、大白卵泡和次级卵泡闭锁率显著增加(P<0.05);血清中黄体生成素和孕酮的含量均呈现降低趋势,但差异不显著(P>0.05);随着周龄增大,蛋鸡机体氧化损伤加剧,表现为肝脏、卵巢中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性均显著降低(P<0.05),丙二醛(MDA)水平显著升高(P<0.05)。结果表明,蛋鸡卵巢机能衰退可能与体内(肝脏、卵巢和血清)氧化损伤加重有关。     

Promotion of ovarian follicular development by injecting vascular endothelial growth factor (VEGF) and growth differentiation factor 9 (GDF-9) genes

Ovarian follicular development in mammals is the complex process including endocrine, paracrine and autocrine. There is the development of four basic stages of ovarian follicles, i.e. the primordial, primary, secondary and tertiary or Graafian follicles. There are few blood vessels in the cortical area where primordial and primary follicles are assembled. The development of these follicles is stimulated by oocytes derived factor including growth differentiation factor 9 (GDF-9) or bone morphogenetic protein 15 (BMP-15). Porcine GDF-9 complementary DNA (cDNA) cloned, and then injected its gene into the ovary in gilts. The injection of porcine GDF-9 gene resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles, indicating that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary. On the other hand, the development of antral follicles is associated with increased density of blood vessels within the theca cell layers surrounding the follicles. A recent study reported that vascular endothelial growth factor (VEGF) play an important role in the process of thecal angiogenesis during follicular development. To investigate whether additional induction of thecal angiogenesis would support subsequent follicular development, miniature gilts were directly injected VEGF gene into the ovary. Injection of VEGF gene increased the levels of mRNA expression of VEGF 120 and VEGF 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene compared with those treated with eCG alone, indicating that the regulation of thecal angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. This technique may be an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.     

Colour Doppler sonography of cystic ovarian follicles in cows

The goals of the present study were to investigate whether colour Doppler sonography can be used to differentiate temporary from persistent ovarian follicles and follicles with luteal tissue from follicles without luteal tissue and to assess the response of follicular cysts to administration of a gonadotropin releasing hormone (GnRH) analogue. Fifty-four cows having ovarian follicular structures with a diameter of >15 mm but no corpus luteum were included. These cows were examined via B-mode and colour Doppler sonography. The same examinations were repeated 10 to 12 days later, and the cows with follicular cysts (n=17) received a GnRH analogue. Blood flow was measured before and 30 min after treatment. Ten to 12 days later, the response to treatment was assessed using B-mode sonography. While 31 of 54 follicles disappeared spontaneously (temporary follicles), 23 follicles persisted and were diagnosed as cystic ovarian follicles (COFs). There was no difference between temporary follicles and COFs in regard to total area, wall thickness or the perfused area. In the luteinized follicles (n=13), based on the plasma progesterone concentration, total area was twice as large, wall thickness was three times greater and the perfused area was 4.5 times larger than those of the non-luteinized follicles (n=41). The sensitivity of diagnosing luteinized follicles was 61.5% using B-mode sonography and 92.3% using colour Doppler sonography. Twelve cows responded to GnRH, and five cows did not. There was a trend (P=0.07) toward higher (59.3%) blood flow in the cyst wall 30 min after treatment in the responding cows compared with the non-responding cows. Our results showed that the perfused area more accurately reflects active luteal tissue than wall thickness. Thus, colour Doppler sonography is superior to B-mode sonography for differentiating follicular and luteal cysts and aids in the selection of treatment. However, exact prediction of COFs destined to regress or persist and the response of COFs to treatment with a GnRH analogue were not possible using colour Doppler sonography.     

The asymmetric distribution of ovarian functional structures in cattle

The results of continually repeated transrectal palpations, performed in 168 post partum periods, 383 estrous cycles and 178 early pregnancies, were used to describe and to discuss the left-right distribution of ovarian functional structures. In cycling as well as in pregnant cattle, anovulatory interestral follicles and estrous follicles or corpora lutea in the mean were all distributed at 41% and 59% on the left and right ovaries respectively. No signs were found indicating that the position of functional structures would be influenced by local interactions between follicles and corpora lutea. At least for cyclic ovarian activity, and in early pregnancy, the interrelationship observed between the locations of these structures could be put down to the normally increased activity of the right ovary. After delivery, the first follicles preferentially became discernible on the ovary opposite to the previously pregnant uterine horn. But, as from the 4th follicle p.p. onward, the distribution of new ovarian structures again agreed with the one of the ensuing reproductive stages. After calving, probably the position of new follicles is temporally influenced by direct signals from the uterine horns affected differently by pregnancy. Several observations indicate that the factors causing asymmetrical ovarian activity could exert a selective effect on the recruitment of the dominant and solitary interestral follicles from the pool of their minor and less differentiated precursors.     

Three-dimensional reconstruction of the equine ovary

The equine ovary has a very unique structure in terms of its extreme large size, the presence of the ovulation fossa and the inverted location of its cortex and medulla. In the previous study, it was recognized that the application of three-dimensional internal structure microscopy (3D-ISM) to observe the mare ovary is very effective. Three-dimensional reconstruction of serially sliced images made by 3D-ISM was successful in this study with the aid of the sophisticated image processing technique. The rotation of the reconstructed ovary has been carried out with and without the application of the transparency technique in the ovarian stromal region. The spatial localization of follicles and corpus luteum was clearly visualized by rotating the reconstructed image of the ovary. The extraction of the images of follicles and corpus luteum was also available and gave a quantifiable understanding of their structure.     

Follicle formation in the canine ovary after autografting to a peripheral site

This study reports about follicular development on the surface of canine ovarian tissue after autografting under the fascia of the thoracolumbar muscle and about meiotic resumption of follicle-derived oocyte after maturation culture. After ovarian excision from a bitch, each ovary of the pairs was cut approximately into half. The hemi-ovaries were transplanted into the bitch of origin at three different body sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle, and in the deltoid muscle in the scapular region). All grafted ovaries were recovered from the bitch at 35 days post-transplantation. A visible antral follicle was observed on the surface of the ovary grafted under the thoracolumbar fascia. Histological examination revealed viable follicles at different stages of development irrespective of graft site. Most granulosa cells in the follicles at different stages of development expressed proliferating cell nuclear antigen (PCNA). A total of three oocytes were collected from an ovary grafted under the fascia of the thoracolumbar muscle, wherein an oocyte reached metaphase I after maturation culture. This is the first report to demonstrate follicular development and meiotic resumption of oocytes recovered from autografted canine ovarian tissues.     

Expression of aquaporin 4 in the chicken ovary in relation to follicle development

In the mammalian ovary, aquaporins (AQPs) are thought to be involved in the regulation of fluid transport within the follicular wall and antrum formation. Data concerning the AQPs in the avian ovary is very limited. Therefore, the present study was designed to examine whether the AQP4 is present in the chicken ovary, and if so, what is its distribution in the ovarian compartment of the laying hen. Localization of AQP4 in the ovarian follicles at different stage of development was also investigated. After decapitation of hens the stroma with primordial follicles and white (1–4 mm), yellowish (4–8 mm), small yellow and the three largest yellow pre‐ovulatory follicles F3‐F1 (F3 < F2 < F1; 20–36 mm) were isolated from the ovary. The granulosa and theca layers were separated from the pre‐ovulatory follicles. The AQP4 mRNA and protein were detected in all examined ovarian compartments by the real‐time PCR and Western blot analyses, respectively. The relative expression of AQP4 was depended on follicular size and the layer of follicular wall. It was the lowest in the granulosa layer of pre‐ovulatory follicles and the highest in the ovarian stroma as well as white and yellowish follicles. Along with approaching of the largest follicle to ovulation the gradual decrease in AQP4 protein level in the granulosa layer was observed. Immunoreactivity for AQP4 was present in the granulosa and theca cells (theca interna ≥ theca externa > granulosa). The obtained results suggest that AQP4 may take part in the regulation of water transport required for follicle development in the chicken ovary.     

Relationship between ovarian weight and follicular population in heifers

The size of the ovary varies substantially among cattle. This variation may influence the potential of the ovary to produce follicles. In the present study, we examined whether a relationship exists between the weight of the ovary and the number of antral follicles >or=1 mm. Paired ovaries were obtained from Holstein x Japanese Black F1 heifers. Follicles were classified into three size categories (small: 1.0-<5.0 mm, medium: 5.0-<8.5 mm and large: >or=8.5 mm), and the number of follicles in each category was recorded. Large variations in the weight of ovaries and the number of follicles were observed among animals. Significant positive correlations (r>or=0.4, P<0.001) were found between the weight of intact ovaries and the number of follicles in all three categories for the ovary contralateral to CL (OCC) and in the small follicles for the ovary ipsilateral to CL (OIC). Significant positive correlations (r>0.4, P<0.0001) were also observed between the weight of ovaries devoid of CL and follicles and the number of small and medium follicles in both OIC and OCC, indicating that the correlation is not due to the increase in ovarian weight associated with the increase in follicular number. Paired ovaries contained a similar number of small and medium follicles, and significant positive correlations were observed between them (r>0.6, P<0.0001). There were significant positive correlations between the weight of OCC and the number of small and medium follicles in paired ovaries (r>0.4, P<0.0001). These results suggest that 1) the weight of an ovary reflects the potential of the ovary to produce antral follicles, and 2) a rough estimation of follicular population might be possible by using the weight of the ovary contralateral to CL in heifers.     

Follicular development in cattle; changes in their count and size during the ovarian cycle

Follicular development was examined by transrectal ultrasound scanning in 12 heifers during 51 oestrous cycles. Internal diameters of largest and second largest follicles and the number of smaller ovarian vesicles were determined. Diameters of dominant follicles showed inverse growth pattern to the second largest follicles and numbers of smaller follicles (greater than or equal to 5 mm). There was an increase in diameters of the largest follicles from beginning of dioestrous to day 9 and from time of luteolysis to ovulation, which was coincident which a decrease in diameters of the second largest follicles and numbers of smaller ovarian vesicles. Smaller follicles increased in count and diameter, when the dominant follicle achieved its largest dimension and started to regress. The cyclic corpus luteum had no local influence on diameters of the largest and second largest follicles in the ovary bearing the corpus luteum versus the contralateral ovary. Internal diameters of oestrous follicles measured 14.7 +/- 2.6 mm in heifers and 15.3 +/- 2.9 mm in cows at the day of oestrous (p greater than 0.05; t-test). Dioestrous follicles with similar size were detected during various stages of the oestrous cycle. The diameter of the dominant follicle is not an accurate criterion for determining the stage of the oestrous cycle.     

Mast cells in the ovary of Gallus gallus domesticus

1. Histamine or mast cells are involved in mammalian ovary function. Their role in the avian ovary is not known. In the present study mast cell distribution in the ovary of the domestic fowl was studied. 2. Mast cells were distributed throughout the ovary, both in the stroma of medullary and cortical regions as well as in the thecae of normal and atretic follicles. In the stroma, mast cells were especially abundant in the area just below the germinal epithelium (GE) and followed the contours of the GE. 3. In the follicles, mast cells were more obvious in the thecae of small non-yolky follicles, whereas they were compressed and scattered in the larger yolky follicles. They were more frequently seen in the theca externa than in the theca interna and in their ultrastructure showed characteristic mast cell granules. 4. Some of the atretic follicles showed increased mast cells in their thecae. Postovulatory follicles had very few mast cells. 5. The possible role of the mast cells in the ovarian activity of domestic fowl is discussed.