The present investigation was carried out to assess comparative efficacy of transvaginal ultrasound guided ablation of cystic follicle at field level with progesterone based hormonal therapy for treating ovarian cyst. Thirty dairy cattle suffering from cystic follicle were divided randomly in two groups viz. cyst ablation group (n=15) and hormonally treated group (n=15). 8 cows showing normal estrus with duration of 1-2 days were also selected. Following cyst ablation, most of the cows (12/15; 80%) showed spontaneous estrus of normal duration within 9-33 days. Mean duration from cyst ablation to spontaneous estrus was 21.75±2.04 days. Following CIDR removal and PGF2α administration, 86.66% (13/15) cows exhibited estrus between 24-96 hrs followed by ovulation between 12-48 hrs. There were significant (P<0.05) higher concentrations of progesterone and estradiol in cystic follicular fluid as compared to those in normal preovulatory follicular fluid. The pregnancy rate turned out to be 33.33% and 38.46%, respectively, in ablated versus hormonally treated cattle. So, transvaginal ultrasound guided cyst ablation can be effectively used as an alternative non-hormonal method for treating cystic ovarian follicles in dairy cattle.
Cystic ovarian follicles (COF) lead to prolonged calving to conception interval, reduced milk production and increased culling rate which aggravates the economic loss to the farmers. It has been estimated to add between 22 to 64 additional days open and reduced milk production (Silvia et al., 2002). Following spontaneous regression, the recurrence of new cyst is about 57% (Cook et al., 1990). Following understanding of its etiology, predisposing factors and diagnosis, a timely intervention is often required. The treatment of cystic ovary involves use of GnRH, hCG and progesterone but with variable outcomes (Kim et al., 2006; Singh et al., 2011; Honparkhe et al., 2011; Singh et al., 2012). Alternatively, emptying of the cystic fluid may be advantageous in such cases (Cairoli et al., 2002). In this regard, transvaginal-guided needle aspiration (Lievaart et al., 2006) or transvaginal ultrasound guided physical ablation (Amiridis, 2009) of ovarian cysts have been considered safe as compared to manual rupture of cyst during trans-rectal palpation. The technique has been proved to be a reliable and efficient to obtain oocytes (Ratto et al., 2011), for induction of new follicular wave (Martinez et al., 2000; Honparkhe et al., 2014) and follicular fluid collection (de Castro e Paula et al., 2008; Shehab- El-Deen et al., 2010). The repeated transvaginal ultrasound guided follicle ablation to induce wave emergence can be used in cows and buffaloes with no harmful effects on conception rate (Rao et al., 2009; Aller et al., 2010). It was hypothesized that transvaginal ultrasound guided ovarian cyst ablation could be a treatment option apart from conventional hormonal therapies. So, the present study was planned with the objective to assess comparative efficacy of transvaginal ultrasound guided cyst ablation with hormonal therapy for treating ovarian cysts.
Materials and Methods
Selection of Animals
The animals suffering from cystic ovarian follicle (n=30) were taken from field and the control animals (n=8) were taken from the university dairy farm for the study. The cystic cows had history of nymphomania, irregular prolonged estrus, aggressive behavior, and breeding (natural/AI) more than three times. These cows were suffering from the condition from last 6 months to 4 yrs, hence considered delayed cases. Additionally, the data regarding parity, body condition score and milk yield was also recorded.
We propose that cystic ovarian follicle be defined as follicle that had diameter of ≥ 25 mm and wall thickness of < 3mm (Fig. 1). This was confirmed by transrectal B-mode ultrasonography (Agroscan, 5/7.5 MHz transducer) twice at 10 days interval. Follicular fluid (normal and cystic) progesterone using progesterone antisera raised in the Department of Veterinary Gynaecology and Obstetrics, GADVASU, Ludhiana (Ghuman et al., 2009) was estimated by liquid phase Radioimmunoassay (RIA). The sensitivity of assay was 0.1 ng/ml, with mean intra- and inter-assay coefficients of variance to be 6.0 and 9.3 percent, respectively. Estradiol concentration was estimated using pre-coated Enzyme linked immune sorbent assay (ELISA) plate’s specific for bovine (Shanghai Crystal Day Biotech Co, Shanghai, China). The sensitivity of assay was 2.53 pg/ml with mean intra-assay coefficient of variation was 2.43 percent. This study was reviewed, and approved by the Institutional Animal Ethics Committee (IAEC) and conducted according to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi. The reference number was GADVASU/2014/IAEC/22/01805.
Fig. 1: (Left) showing the maximum diameter of cystic ovarian follicle (average of the horizontal and vertical diameter at the widest point) measured using the in-built electronic callipers; (Right) showing transvaginal ultrasound guided needle (arrow) advancing in COF
Group 1 (Ablation Group; n=15)
In this group (n=8) normal preovulatory dominant follicle (≥ 14 mm) and (n=15) cystic ovarian follicles were ablated through transvaginal ultrasound guided ablation (Fig. 1). In cystic cows ovarian ultrasound was done at weekly interval till day 21. These cows were allowed to be inseminated after 21 days period of examination only at spontaneous estrus. The normal and cystic follicular fluid collected was centrifuged and stored at -20°c till analysis.
Group 2 (Hormone Group; n=15)
The cows of this group (n=15) were subjected to CIDR based Ovsynch protocol i.e. insertion of a CIDR (controlled internal drug release) device containing progesterone (1.38 g) + GnRH analogue (20 µg Buserelin acetate, Inj. Receptal, i.m.) on day 0, PGF2α analogue (cloprostenol, 500μg Inj. Estrumate, i.m.) at the time of CIDR removal on day 7, GnRH (20 µg Buserelin acetate, Inj. Receptal; i.m) on day 9 and AI on day 10/11.
About 10 ml of blood samples from all the cows were collected through jugular vein in heparinised vials from the start till the end of experiment as on day 0, 7, 14 and 21 following ablation and day 0, 7, 9 and 10/11 following hormonal treatment. Plasma was separated out and stored at -20°C until assay.
The data was expressed as Mean ± SEM, statistical analysis was conducted using IBM SPSS statistics 21.0 windows. Pregnancy rates were compared in both the groups (ablation and hormonal) using chi-square test.
Results and Discussion
Comparative Efficacy of Transvaginal Ultrasound Guided Ablation with Hormones
In the present study, following cyst ablation, most of the cows (80%) showed spontaneous estrus of normal duration within 9-33 days (Table 1). Only, three cows (20%) did not respond to the treatment and the recurrence of cyst was observed in these cows.
Table 1: Response of Transvaginal ablation (Gp 1) and CIDR based Ovsynch protocol (Gp 2)
|Parameters||Ablation||CIDR based Ovsynch|
|Recovery (%) w.r.t cyst formation||12/15 (80%)||13/15 (86.66)|
|Recurrence (%) w.r.t cyst formation||3/15 (20%)||2/15 (13.33)|
|Days to spontaneous estrus (n=12)||21.75±2.04 (9-33 d)||–|
|Interval of PGF2α to estrus (hrs)||–||2.57±0.25 (24-96)|
|Interval of estrus to ovulation (hrs)||–||35.07±3.45 (12-48)|
|Interval of PGF2α to ovulation (hrs)||–||3.87±0.20 (60-108)|
|Size of ovulatory follicle (mm)||–||14.9±0.77 (10-20)|
|Pregnancy rate (%)||4/12 (33.33%)||5/13 (38.46%)|
Cairoli et al. (2002) also reported 75.6% cows in regular cyclicity within 30 days following ablation. However, Amiridis (2009) reported 94.4% recovery following cyst aspiration in cows with COF (40-60 days postpartum). The mean duration from cyst ablation to spontaneous estrus was 21.75±2.04 days in the present study that was higher than that observed by Amirids (2009). Following CIDR removal and PGF2α administration, 86.66% cows showed regularity in estrus cycle followed by ovulation in next 12-48 hrs. Size of ovulatory follicle was 14.9±0.77 mm. Crane et al. (2006) also reported 78.7% animals in estrus with ovulatory follicle size of 16.9±0.87. Also, in previous study by Todoroki et al. (2001), within 3 days after CIDR removal, estrous behaviour was detected followed by ovulation of the dominant follicle. In contrary, this recurrence was higher (5.5%, P < 0.05) as compared to Amiridis (2009). Lower recovery could be due to most of the delayed cases (6 months to >3 years) suffering from COF that were included in the present study. The pregnancy rates (in cows recovered from cyst) in Group 1 and 2 were 33.33% and 38.46%, respectively. Amiridis (2009) also recorded pregnancy rate of 33.3% following ablation on 40-58 days post-partum (pp) cows suffering from cystic follicle. However, Kim et al. (2006) observed 52.3% (P < 0.05) conception rate after the CIDR-based TAI protocol that was inconsistent to our study.
Follicular Fluid Hormonal Concentrations Following Treatment
Transvaginal ultrasound guided ablation of cystic ovarian follicle and normal preovulatory follicles was done and the progesterone (by RIA) and estradiol (by ELISA) concentrations were measured in follicular fluid. In the present study, there were significant (Table2; P<0.05) higher concentrations of progesterone and estradiol in cystic follicular fluid as compared to those in normal preovulatory follicular fluid (18.88±2.84 ng /ml and 626.66±136.2 pg/ml vs 7.13±2.17 ng/ml and 215.75±16.04 pg/ml respectively). In the present study follicular fluid hormonal concentrations were in accordance with previous studies (Maniwa et al., 2005; Khan et al., 2011) those revealed higher concentrations of P4 and E2 in cystic follicular fluids causing defective endocrine milieu followed by cyst development. Grado-Ahuir et al. (2011) also showed greater (P<0.05) progesterone and estradiol (441.5±75.7 ng/ml and 405.8±72.8 ng/ml) in cystic than that in normal preovulatory follicular fluids (163.8±76.1 ng/ml and 322.6±75.6 ng/ml). According to Takagi et al. (1998), progesterone and estradiol varied in range from 7.3-289.4 and 0.01-51.5 ng/ml, respectively. The variations in the steroid hormone concentrations in different studies might be attributed to dissimilar steroidogenic capacity of cyst as many intermediate forms exist with limited or extensive luteinization of the follicle wall. Roth et al. (2012) classified ovarian cyst on the basis of follicular fluid hormone concentrations as estradiol dominant cyst (E2; 284-659ng/ml), progesterone dominant cyst (P4; 586-3288 ng/ml) and low steroidogenic cyst (low E2; 23-61 ng/ml, low P4; 17-205 ng/ml) and suggested that low peripheral P4 play role in the persistence of follicle and development of cyst.
Table 2: Mean ± SEM progesterone and estradiol concentrations in cystic and normal preovulatory follicular fluid (FF), Mean ± S.E.M progesterone (P4; ng/ml) and estrogen (E2 pg/ml) concentration in blood plasma on different days following treatment
|End points||Ablation||CIDR based Ovsynch protocol|
|P4 (FF)||E2 (FF)||P4 (FF)||E2 (FF)|
|Cystic ovary (n=15)||18.88±2.84A||626.66±136.2A||–||–|
|Normal ovary (n=8)||7.13±2.17B||215.75±16.04B||–||–|
|Days following treatment||P4 (Plasma)||E2 (Plasma)||P4 (Plasma)||E2 (Plasma)|
A-B, a-bDifferent superscripts within a column are significantly different (P < 0.05)
Blood Plasma Hormonal Concentrations Following Treatment
Following ablation, significant decrease was observed in progesterone concentration on day 7 and 14 with slight increase at day 21 (Table 2; P<0.05). Following CIDR based ovsynch protocol, significant decrease in progesterone concentration from 3.21± 0.39 ng/ml at day 0 to 0.59±0.44 ng/ml at the end of experiment was observed. Estradiol concentration was also significantly reduced (P<0.05) from 612.66±124.20 pg/ml at day 0 to 271.66±88.48 pg/ml until the end of the experiment.
In the present study blood plasma hormonal concentrations were in accordance with results obtained by Todoroki et al. (2011); Roth et al. (2012). Progesterone concentrations were statistically higher in the cows with follicular cysts compared to normal cows exhibiting regular cyclicity. Comparing this data with Ortegon et al. (2006), a significant (P<0.05) drop in P4 concentration following aspiration (from 3.26 ± 1.5 to 2.16 ± 0.1 nmol/L) was observed as our study. Abnormal circulating progesterone concentrations interfere with hypothalamic responsiveness to estradiol and result in the formation of cysts. 66% of cows with cysts and no detectable luteal tissue in cyst or ovary, had intermediate circulating progesterone concentrations (0.1-1 ng/ml), and only 10% of the newly-developed follicles ovulated in these cows (Hatler et al., 2003; Brito and Palmer 2004). It may be possibly due to luteinization of a previous preovulatory follicle and follicles that develop in the presence of this progesterone are likely to become cysts themselves. After removal of the cystic content the cows are deprived from the main source of oestrogens along with other possible locally acting factors that leads to a new follicle development and finally ovulation (Amiridis, 2009). Thus, regression of the cyst led to restoration of the normal function of the hypothalamic–pituitary–ovarian axis. It could be better achieved by transvaginal ablation in dairy cows suffering from prolonged cystic follicles. So, transvaginal ablation can be effectively used as an alternative to hormones at field level for prolonged duration cystic ovarian follicles.