Evaluation of tubal patency with hysterosalpingo-contrast sonography using sodium alginate solution and the subsequent fertility outcomes in repeat breeder cattle

Abstract

Tubal abnormalities were evaluated on hysterosalpingo-contrast sonography (HyCoSy) using sodium alginate solution as a contrast agent in 18 repeat breeder cattle. Clear contrast enhancement was observed from the uterine horn to the tubal infundibulum in the patent cases, and the percentage of cases with unilateral or bilateral passage disorder was 64.7%. The post-examination artificial insemination conception rate when ovulatory follicles were present on the patent side was 64.3%, which was significantly higher than 12.5% seen when ovulatory follicles were present on the side affected by passage disorder (P < 0.05). The polymorphonuclear leukocyte percentage in the recovered contrast agent was significantly higher on the passage disorder side than on the patent side, whereas there was no significant difference in C-reactive protein concentrations between the patent and passage disorder sides. The present method can be used for diagnosing tubal abnormalities, and chronic inflammatory changes may be related to tubal passage disorder.

Repeat breeder syndrome, in which attempts to achieve conception in cattle fail despite more than three insemination cycles being performed and the absence of apparent clinical abnormalities, is a major factor that reduces productivity in the dairy and beef industries [1]. One of the causes of repeat breeder syndrome is tubal passage disorder [2]. Previous studies found tubal passage disorder in various percentages of repeat breeder cows; i.e., 29.8% and 44% of repeat breeder cows exhibited tubal obstruction in post-slaughter examinations [2] and a dye-based tubal patency test [3], respectively.

Several diagnostic examinations; i.e., the tubal ventilation test [4, 5], starch test [6], and phenolsulphonphthalein (PSP) test [3], have been reported as methods for detecting tubal passage disorders. However, these are not widely used in clinical practice because they are not technically simple enough for veterinarians. The tubal ventilation test requires complicated instruments to be prepared, such as a gas injection regulator and kymograph to monitor the internal pressure of the reproductive tract. The starch and PSP tests require several rounds of treatment/examination, and it takes several days to obtain the results. On the other hand, hysterosalpingo-contrast sonography (HyCoSy) is one of the most frequently used methods for detecting tubal patency in humans [7, 8]. This technique has a lot of advantages, such as its ability to obtain diagnostic results with minimal invasiveness in a short period of time on the same day [9]. Recently, performing HyCoSy using human medical contrast agent was shown to be clinically useful for diagnosing tubal disorders in cows [10, 11].

Sodium alginate has a wide range of applications as a food additive [12], e.g., as a thickener and stabilizer; a pharmaceutical agent, e.g., as a gastric mucous membrane protectant [13], a hemostatic agent [14]; an obstetric lubricant in the livestock industry; and a feed additive [15], and is highly safe [16] and inexpensive. In addition, sodium alginate becomes a viscous liquid when it is made into an aqueous solution, which increases the retention time of microbubbles, suggesting that it may be effective as an ultrasound contrast agent [17]. The use of this agent, which is extremely cheap and versatile compared with conventional human contrast agents, would strongly encourage the use of HyCoSy as a routine test for tubal disorders in cattle in the field of veterinary medicine.

The aims of the present study were to determine 1) the usefulness of using sodium alginate solution to generate microbubbles (sodium alginate solution microbubbles; SAM) as an ultrasound contrast agent for diagnosing tubal passage disorders in HyCoSy examinations, and 2) the relationships between tubal passage disorders and clinical indices; i.e., inflammatory markers (the polymorphonuclear leukocyte percentage (PMN%) and C-reactive protein (CRP) levels) or subsequent fertility outcomes in repeat breeder cattle.

In the patent cases of the present study, the injected contrast agent filled the lumen of the uterine horn, and the oviduct was visualized as lines and circles on longitudinal and transverse sections, respectively. The oviduct became tortuous toward to the ovary, and the tubal infundibulum was identified as a highly echogenic structure bordering the ovary (Figs. 1A–D).

Fig. 1.

Contrast enhancement of the uterus and oviducts produced by using SAM as a contrast agent in the patent cases; (A) A cross-section of the uterine horn filled with SAM is shown (arrow). (B) Oviducts are visualized as lines (arrow) in longitudinal sections. (C) Numerous sections of oviducts are seen at once (arrow) as one approaches the ovary. (D) The tubal infundibulum is identified as a highly echogenic structure (arrow) in close proximity to the ovary. OV: Ovary.

HyCoSy was performed on a total of 35 oviducts in 18 cows/heifers. There was one oviduct (#80, right) that could not be subjected to HyCoSy due to the difficulty of catheter insertion. The contrast-enhancement findings were classified into two categories, patent and passage disorder. They were further categorized into three patterns, as shown in Supplementary Table 1. The overall results of the tubal patency test based on the abovementioned contrast patterns were as follows: 6 cows were classified as bilaterally patent (35.3%), 9 cows were classified as having unilateral passage disorder (52.9%), and 2 cows were classified as having bilateral passage disorder (11.8%). One cow could not be diagnosed. The bacteriological test was positive in 5 out of 18 cows, and urovagina was found with vaginoscopy in 2 cows.

The results of the tubal patency examinations and conception outcomes are summarized in Table 1. The conception rate for artificial insemination (AI) when an ovulatory follicle was present on the patent side was 64.3% (9/14), which was significantly higher than the 12.5% (1/8) for AI conducted on a side affected by passage disorder (P < 0.05). Eight out of 9 cows that conceived after AI was performed when an ovulating follicle was present on the patent side conceived after the first post-examination round of AI conducted on the patent side. The conception rate of embryo transfer (ET) in the 4 cases in which it was performed was 50%.

Table 1. The clinical findings of hysterosalpingo-contrast sonography (HyCoSy), bacteriological testing, and post-examination fertility outcomes.

	Clinical findings					Fertility outcomes
Repeat breeder ID	HyCoSy		Bacteriological test (CFU/ml)		Remarks	AI				ET
	Left oviduct	Right oviduct	Left	Right		Patent side		Passage disorder side		Rounds	Conception
						AI rounds	Conception	AI rounds	Conception
#8792	◯ 1)	× 2)
#8800	◯	◯				1	+ 4)			1	−
#9929	×	◯
#3381	◯	×		CNS (75)		2	− 5)	2	−
#3	◯	◯	Trueperella (150)	Streptococcus spp. (50)		2	−
#1503	×	◯		Streptococcus spp. (50)		1	+
#0798	◯	◯				1	+
#9113	◯	×				1	+	1	−
#3323	×	×						3	+
#0810	◯	×			Urovagina			1	−	1	−
#2107	◯	×						1	−	1	+
#802	◯	×				2	+
#33	◯	◯				1	+
#0850	◯	◯				1	+
#23	×	×		Trueperella (100)	Urovagina
#80	×	# 3)								1	+
#0273	◯	◯				1	+
#5757	◯	×	Corynebacterium (100)	Streptococcus spp. (50)		1	+
Total						14	9	8	1	4	2
Conception rate (%)						64.3 *		12.5 *		50.0

* P < 0.05 for the difference between values, 1) patent, 2) passage disorder, 3) not performed, 4) conception, 5) no conception.

The relationships between the results of the tubal patency examination and the PMN% or CRP concentration in the collected fluid are shown in Fig. 2. PMN% was significantly higher on the side affected by passage disorder (Median [interquartile range (IQR)] = 0.3% [0.1–1.0]) than on the patent side (0% [0.0–0.0]) (P < 0.01) (Fig. 2a). CRP concentrations were lower than the previously reported diagnostic criteria (20 ng/ml) for uterine inflammation [18] on both the patent side (3.27 ng/ml [2.43–4.37]) and the side affected by passage disorder (4.17 ng/ml [2.43–11.53]), with no significant difference between them (P = 0.56) (Fig. 2b).

Fig. 2.

PMN% (a) and CRP concentration (b) in intrauterine perfusate from patent oviducts and oviducts affected by passage disorder. (a) PMN% was significantly higher on the side affected by passage disorder (Median [IQR] = 0.3% [0.1–1.0]) than on the patent side (0% [0.0–0.0]) (P < 0.01). (b) CRP concentrations were not significantly different between the patent side (3.27 ng/ml [2.43–4.37]) and the side affected by passage disorder (4.17 ng/ml [2.43–11.53]) (P = 0.56). White circles (○) indicate outliers, defined as values more than 1.5 times the quartile range away from the first or third quartile value.

HyCoSy with SAM produced clear contrast enhancement from the uterine horn to the tubal infundibulum, which was similar to previous reports [10, 11] in which an ultrasound contrast agent for humans was used in cattle. The present results indicate that SAM could be utilized as a contrast agent for HyCoSy in cattle. Using this method, patent cases may be diagnosed relatively easily because the oviduct was clearly imaged from the uterus to the ovary. On the other hand, further investigation is needed to determine its diagnostic accuracy for passage disorder because various technical limitations may influence the diagnosis of the condition. In fact, one cow that was diagnosed with bilateral tubal passage disorder conceived after the subsequent AI. In this case, HyCoSy was performed with a volume of 30 ml as previously reported [10, 11], which may be insufficient for animals with large uterine horn volumes, and hence, it is possible that the contrast agent did not reach the depth of the oviduct. For HyCoSy in Holstein cows, an injection of approximately 50 ml or more of contrast medium might be appropriate. Moreover, the percentage of repeat breeder cows with unilateral or bilateral passage disorder in this study was 64.7%, which is higher than the previously reported rates of 29.8% [2] and 44% [3]. The reason for the difference between the present and previous studies is unclear, although it may have been due to the accuracy of passage disorder diagnosis, which may be affected by appropriate amount of contrast medium and the characteristics of individual cases.

Based on the examinations, the possible causes of repeat breeding in the present cases were suggested to include subclinical uterine infection, urovagina, and tubal passage disorder. These findings indicate the clinical importance of detailed medical examinations for identifying the cause of repeat breeding in cattle. Regarding tubal examinations, most of the previous reports on the use of HyCoSy in cows focused only on the imaging findings of patent cases. In this study, we also examined the imaging findings of cases of passage disorder. Based on the diagnoses made in the present study, a significant difference in conception rates after AI was found between the ovulatory follicles on the patent side and those on the side affected by passage disorder. Of the five cows that were diagnosed with bacteriological infections, 2 became pregnant (#1503, #5757) after the first post-examination round of AI in which an ovulatory follicle was present on the patent side. These findings support those of previous studies that found that tubal passage disorder is one of the major causes of repeat breeding in cattle [2, 3]. Moreover, AI conception rate after HyCoSy for repeat breeder cows was 45.5% (10/22), implying that HyCoSy itself has a fertility-enhancing effect. Uterine irrigation of repeat breeder cows has been shown to increase their fertility [19]. Since HyCoSy involves a similar procedure to uterine irrigation, it is possible that the same mechanism may be involved in the observed increase in fertility. Uterine irrigation is thought to wash away dirt in the uterus, improve the environment [20], and eliminate tubal passage disorders through the effects of pressure [21]. The findings of P2 and P3 of the HyCoSy category shown in Supplementary Table 1 possibly reflect a situation in which the oviducts that had been impaired in transit were restored to passage by the procedure. In fact, conception was observed 2 out of 2 times in AI after HyCoSy in oviducts that showed a P2 pattern.

The PMN% of the recovered fluid was significantly higher on the side affected by passage disorder than on the patent side. The PMN% is commonly used as a diagnostic indicator of inflammation in the endometria of cows [22], suggesting that inflammatory lesions may have arisen in the endometrium and/or tubal epithelium on the sides affected by passage disorder. The PMN% was less than 3–5%, which is usually considered the cut-off value for endometritis [23, 24], on the passage disorder side in the present cases. It is possible that the amount of PMN in the recovered fluid was estimated to be low in the case of inflammation confined to the oviduct because the amount of contrast agent perfused into the oviduct was small. To the best our knowledge, no previous studies have reported PMN% data for live cattle with salpingitis; therefore, it is unclear whether the PMN% values on the passage disorder side specifically reflect tubal inflammation in the current methodological setting.

Our previous studies have reported that the CRP concentration in uterine lavage samples is a useful indicator of acute local inflammation in the uterus. A correlation was found between the concentration of CRP in uterine lavage fluid and PMN% at 3 and 6 weeks postpartum [25], and uterine CRP production was increased by experimentally induced inflammation on the day following the intrauterine infusion of iodine [18]. In this study, while differences in PMN% were detected between the patent and passage disorder sides, no significant differences in CRP concentrations were observed, with low values being seen on both the patent and passage disorder sides. One possible interpretation of this discrepancy is that the cases of passage disorder in this study may have involved chronic, rather than acute, inflammation. It has been noted that endometritis results from infectious inflammation caused in the early postpartum period [26] and that the majority of salpingitis is due to spillover from endometritis [27] in cows. Taking these points and the fact that the HyCoSy was performed a long time after calving into consideration, it is possible that the oviduct had transitioned to a chronic inflammatory process by the time of the examination in the present study.

In conclusion, the present study indicated that SAM produce sufficient contrast enhancement in ultrasound examinations of the reproductive tract and that performing HyCoSy with SAM is a practical tool for the diagnosis of tubal passage abnormalities in cattle.

Methods

Eighteen repeat breeder Holstein dairy cattle from 9 farms in Saitama, Japan, with no abnormal estrus cycles, no pregnancy despite at least three AI attempts (mean ± SD: 6.0 ± 1.7, range: 3–8), and no uterine or ovarian abnormalities by rectal palpation and ultrasonography, were used in this study. They had parity values of 0 to 5, the mean number of days postpartum for producing cows (n = 13) was 315.9 ± 64.2 days (mean ± SD), and age of the heifers in months (n = 5) ranged from 17–24 months. The present study was approved by the Committee for the Care and Use of Research Animals of Tokyo University of Agriculture and Technology (KEN-DO-07-08).

HyCoSy was performed during the luteal phase. After disinfecting the vulva, a sterile balloon catheter (16 Fr perforated balloon catheter, Fujihira Industry Co., Ltd., Tokyo, Japan) was inserted into the uterine horn on one side. When the tip of the catheter was approximately 5 cm cranial to the uterine horn bifurcation, 10 ml of air was injected into the balloon. Then, 30–60 ml of contrast agent was injected through the catheter, and the uterus and oviduct were scanned using the B-mode of the ultrasound system. The contrast agent, SAM, was made by dissolving 500 mg of a sterilized sodium alginate preparation (Alto powder, Kaigen Pharma Co., Ltd., Osaka, Japan) in 80 to 100 ml of sterilized saline solution, adjusting the concentration to 0.5 to 0.625%, and then generating microbubbles. Microbubbles were generated by aspirating sodium alginate solution and air at a ratio of 5:1 into a syringe of at least 50 ml capacity, connecting it to a syringe of the same capacity with tubing, and transferring the solution between the syringes 50 times. An HS-101V (Honda electronics Co., Ltd., Tokyo, Japan) or CTS-800 (Kyoritsu Seiyaku Co., Tokyo, Japan) was used as the ultrasound system. Ultrasound scanning was performed from the uterine horn to the ovary for approximately 5 min to observe the effects of the contrast agent. Then, the findings were classified into two patterns based on the presence or absence of contrast enhancement of the tubal infundibulum: a patent pattern and a passage disorder pattern. After the observation period, the same procedure was performed on the opposite side.

After the HyCoSy, as much contrast agent as possible was removed through a balloon catheter, and the collected fluid was tested for bacteriological contamination (n = 35) and to assess the PMN% (n = 25) and CRP (n = 21) level in some trials. The bacteriological examination was conducted by performing an aerobic culture on blood agar for 24–48 h, before the number of colony-forming units/ml was calculated, and then simple identification of bacterial species was performed based on colony shape, Gram staining, and catalase testing. After smearing the sediment of the collected fluid on a glass slide, simple Giemsa staining was performed, and the percentage of polymorphonuclear leukocytes per 1000 nucleated cells was calculated. The CRP concentration was measured using the C-reactive protein ELISA kit for bovines (CLOUD-CLONE CORP, Katy, TX, USA), as reported previously [25].

After the examination, AI or ET was performed after the detection of spontaneous estrus until conception or until the owner decided to cull the animal. The ovulatory follicular side (for AI) or the luteal side (for ET) was recorded.

Data were analyzed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [28]. Fisher’s exact test was used to compare fertility rates between the two groups. PMN% and CRP concentrations are presented as medians [IQR] for each group, and Mann-Whitney’s U test was used to identify significant differences in these parameters. P-values of < 0.05 were considered significant.

Conflict of interests

The authors have no conflicts of interest directly related to the content of this article.