Introduction

Bovine ovarian cysts are follicles that fail to ovulate at the time of oestrus. Cystic ovaries is a major cause of infertility and economic loss to the dairy industry, and occurred in the dairy cows with 10-13% (Ganverick, 1999). From the predisposing factors leading to the occurrence of cystic ovarian follicles (COF) are high milk yield, season, stress and negative energy balance (Seguin, 1998; Lopez-Gatius et al., 2002). The absence of the preovulatory surge of GnRH and LH are responsible for the development of COF. This phenomenon may be the result of hypothalamic insensitivity to the estradiol surge caused by inadequate exposure to progesterone (Gumen and Wiltbank, 2002). The relationship between circulating progesterone and COF has been docomented by Hatler et al. (2003). Moreover, the presence of COF is detrimental to fertility and some practitioners may resort to manual rupture of the cystic structures, because this is still a documented form of treatment (Seguin, 1998). Some hormones such as GnRH, hCG, LH and progesterone are frequently used to treat this condition (Thatcher et al., 1993), but the outcomes are variable. Some recent protocols for synchronization of ovulation, commonly referred as ovsynch, followed by timed artificial insemination (AI) performed 16-20 h after the 2 ^nd GnRH injection yields pregnancy rates of about 25% in cows suffering from COF (Fricke and Wiltbank, 1999; Bartolome et al., 2000; Meyer, etal., 2007). The response to treatment with GnRH, PGF2α and GnRH were not fully documented. Diagnosis of a COF was based on the palpation per rectum of an abnormally large (≥20 mm) follicle and no corpus luteum, verified by transrectal ultrasonography (Bartolome et al., 2000). After the first GnRH injection, another existing follicle ovulated in 4 cows, and a new follicle developed in all cows then ovulated in all cows within 48 h of the 2 ^nd GnRH injection, and the development of a CL was confirmed in all cases.

This study aimed to, (a) assess the ovarian and endocrine responses in dairy cows affected with cystic ovarian follicles when a standard ovsynch protocol or coordinated sequence of treatments with GnRH were used in the presence of an intra vaginal progesterone releasing device (CIDR), and (b) determine the pregnancy rates following ovsynch and timed AI in dairy cows affected with cystic ovarian follicles.

Material And Methods

Animals

Classification of cows according to the type of ovarian cysts

Schedule-A

Schedule-B

Blood samples and hormonal assay

Statistical analysis

Results

Schedule-A

The examination revealed 15 cows suffering from cystic ovarian follicles, 7, 3 and 5 cows were diagnosed as having follicular, luteinized and persistent cysts, respectively (Table 1). The progesterone concentration was influenced by the time of sampling (P<0.01) and the interactions between type of cystic ovarian follicle and time of sampling (P<0.01). The progesterone concentration of the three types of cysts is presented in Fig. 1. The concentrations were significantly higher at 1 ^st GnRH injection, then decreased after PGF2α injection (13 day). At the 0 day (day of insemination, the concentrations were not variable between types of cysts, then increased again at 7 day (after insemination).

The ovarian responses to the ovsynch treatment and pregnancy outcome are presented in Table 1. Although none of the cystic ovarian follicles ovulated, by 7 days after GnRH treatment, the luteinized structures resembled a large CL were determined. All the cows developed a new follicle after 1 ^st GnRH injection, from which 57.1%, 33.3% and 40% of follicular, luteinized and persistent cyst were ovulated, respectively. A new dominant follicle developed following 1 ^st GnRH treatment in all cows. After the 2 ^nd GnRH treatment, a total of 100%, 66.7% and 100% of the new follicles in cows with follicular, luteinized or persistent cysts were ovulated.

A new CL was confirmed 7 days after AI. Of the 15 cows that were inseminated, 12 (80%) developed a new CL and 7 (46.7%) were confirmed pregnant. The cows with follicular cysts appeared the highest rate of pregnancy (57.1%) followed by the luteinized and persistent cysts (33.3% and 40%, respectively).

Figure 1

Table 1: The effect of treatment with GnRH and PGF2α (Schedule-A) in dairy cows suffering from follicular, luteinized and persistent ovarian cysts.

Figure 2

Figure 1: Progesterone profiles (Mean +/- SEM) of cows classified as having follicular cyst, luteinized cyst or persistent cyst in schedule-A at the 1 GnRH injection (day – 10), at PGF2? (day – 3 ), at artificial insemination (day 0) and at 7 days after artificial insemination (day 7)

Schedule-B

On the basis of hormonal level (Fig. 2), of 14 cows in this trial, 6 were determined to have a follicular cyst (based on a high estradiol and a low progesterone profiles and the absence of any luteal tissue), 3 cows had a luteinized cysts (based on a low estradiol, and a high progesterone profile, and no luteal tissues) and 5 cows had a large CL cyst-like structure (based on a low estradiol and a high progesterone profiles) were categorized as persistent cysts (Table 2).

Although none of the cystic ovarian follicles ovulated after 1 ^st GnRH injection, 8 of the 14 cows ovulated after existing a new follicles, where 4/6 (66.7%) for follicular and 4/5 (80%) for persistent and 0% for luteinized cysts were ovulated. Additionally, all cows developed new follicles in response to GnRH injection. Following to CIDR removal, 12 of 14 cows ovulated the recruited follicles and developed a CL, where all the cows with follicular and persistent cysts and none with luteinized cysts were ovulated. Of the 14 cows that were inseminated, 8 (57.1%) were confirmed pregnant. The cows with follicular cysts showed the highest rate (66.7%) of pregnancy followed by persistent cysts (60%) with low rate (33.3%) detected for luteinized cysts.

There was marked increase in the concentration of progesterone at the day or one day after GnRH injection or CIDR placement, then decreased at oestrus (2-3 days after CIDR). At the presence of palpable CL, the concentration elevated again. On the other hand, the concentration of estradiol decreased at the day of GnRH injection or CIDR placement but markedly increased one day after removal of CIDR, at oestrus (2-3 days after removal), the concentration was still higher than at the presence of a palpable CL (Fig. 3).

Figure 3

Figure 2: Plasma concentrations (Mean±SEM) of progesterone and estradiol in the pretreatment period in dairy cows suffering from follicular, luteinized and persistent ovarian cysts (schedule-B).

Figure 4

Table 2: The effect of treatment with GnRH and PGF2α with CIDR placement (schedule-B) in dairy cows suffering from follicular, luteinized and persistent ovarian cysts.

The percentages of new developed CL as well as the pregnancy rate were higher in the cows of schedule-B (after 2 ^nd GnRH injection or CIDR removal) compared to these cows in schedule-A (after 2 ^nd GnRH injection alone), as shown in Fig. 4. In the cows affected with the follicular and persistent cysts, the percentages of CL and pregnancy were higher in the schedule-B than schedule-A

Figure 5

Figure 3: Plasma progesterone and estradiol profiles (Mean±SEM) in the cow classified as having follicular cyst in schedule-B. the hormonal profile measured at days related to the GnRH and CIDR placement.

Figure 6

Figure 4: The new developed CL and pregnancy rates, when schedule-A (after 2 GnRH injection) compared to schedule-B (after 2 GnRH injection and CIDR removal) in the cows affected with follicular, luteinized and persistent ovarian cysts.

Discussion

Reproductive efficiency in dairy herds increases by inseminating all the cows shortly after the end of the voluntary waiting period, obtaining high pregnancy rate to first service enhancing embryonic and fetal survival (Pursley et al., 1997; Stevenson et al., 1999). In a previous study [Bartolome et al., 2000], the Ovsynch protocol was effective in cows with ovarian cysts. In the present study, pregnancy rates were higher for cows with ovarian cysts in the GnRH+Ovsynch protocol compared to cows in the Ovsynch protocol. Cows with ovarian cysts continue to have follicular waves (Yoshioka et al., 1996); therefore, the dominant follicle may or may not respond to GnRH treatment [Kesler et al., 1981]. In the GnRH+Ovsynch protocol, cows received two doses of GnRH and may be more likely to have a CL at the time of PGF2a. In the present investigation in which 2 different schedules were used, documented the recovery of cows diagnosed with cystic ovarian follicles. Even though none of the cows ovulated a cystic ovarian follicles, 57.1% of the cows ovulated a follicle other than the cystic ovarian follicles that were present at the time og GnRH injection. Ovulation of a cystic ovarian follicle in response to GnRH treatment does not usually occur and has not been observed in other investigations (Garverick, 1999; Fricke and Wiltbank, 1999; Wiltbank et al., 2002). However, luteinization of the cystic ovarian follicle following GnRH-treatment has been reported (Garverick, 1999; Wiltbank et al., 2002; Stevenson et al., 2007).

In the present study, development of a new follicle following GnRH treatment occurred in all cows in both schedules; this response is well documented for normal cycling cows that are treated with the ovsynch protocol (Pursley et al., 1995, Meyer et al., 2007). The effectiveness of the ovsynch and timed artificial insemination protocol in lactating dairy cows diagnosed with ovarian cysts has been reported by Fricke and Wiltbank (1999); Bartolome et al. (2000), with pregnancy rates of 26.9% and 23.6% (calculated based on a reported conception rate). The ovarian responses to the ovsynch treatment were either partially recorded or not recorded in these reports. By using a protocol similar to that used by the above authors, we have determined that all cows developed a new follicle in response to the 1 ^st GnRH treatment and that a majority of these newly recruited follicles, subsequently ovulated. In the cows that were subjected to ovsynch and timed artificial insemination, a 46.7% (in schedule-A without CIDR) and 57.1% (in schedule-B with CIDR) pregnancy rates were achieved in the present study. The persistence of cystic ovarian follicles for prolonged periods has been recorded previously (Garverick, 1999; Noble et al., 2000), and it is highly unlikely that these have any negative influence on a new follicular development or pregnancy establishment.

Regardless the type of ovarian cyst, a new follicle developed following GnRH treatment in 100% of the cows examined. In a high percentage of cases, the newly developed follicle ovulated either in response to a 2 ^nd GnRH treatments (93.3%; schedule-A) or spontaneously (85.7%; schedule-B) depending upon progesterone withdrawal. Cows with cystic ovarian follicles are known to have high plasma LH concentrations (Garverick, 1999) and increased LH pulse frequency (Calder et al., 1999). Increased LH concentrations favor the development and persistence of large ovarian follicles (Taft et al., 1996), whereas treatment with exogenous progesterone induces atresia of persistent follicle (Manikkam and Rajamahendran, 1997; McDowell et al., 1998). The later approach has been used successfully to reduce LH pulse frequency and induce turnover of cystic ovarian follicles in cattle by placing intra vaginal P4-releasing devices for either 9- (Peter, 1998; Bicalho et al., 2007) or 14- (Todoroki et al., 2001) day periods. The results in this study and previous studies (Calder et al., 1999; Todoroki et al., 2001), showed that the cystic ovarian follicles declined in size following insertion of CIDR. Associated with the insertion of a CIDR device and GnRH injection were an increase in progesterone and a concomitant decline in circulating estradiol concentrations. After 9 days of the CIDR removal, estradiol concentrations increased rapidly and in schedule-B all cows come into oestrus within 3 days, and 12/14 (85.7%) cows ovulated spontaneously.

The results of this study and those of others, we recommended that treatment of cystic ovarian follicles must focus on altering the endocrine milieu, such that it allows recruitment of a new follicle (by using GnRH) and induces the turnover of the cystic ovarian follicle (by using a progesterone device), preferably within an ovsynch-type protocol. The importance of ovsynch protocol in the treatment of cystic ovarian follicles is emphasized by the ability of GnRH given after CIDR withdrawal to induce ovulation of a newly recruited follicle in a follicular cystic cow that failed to ovulate spontaneously after CIDR withdrawal in a previous instance. However, the observations that 100% of the cystic cows developed a new follicle after GnRH injection, and 46.7% (schedule-A) or 57.1% (schedule-B) of the inseminated cows conceived following ovsynch and timed artificial insemination; and in many of these cows; the cystic ovarian follicles persisted with no approach detrimental influence on the establishment or maintenance of pregnancy.

This study concluded that, the cystic cows administered GnRH (day 0), followed by PGF2α (day 7), +/- CIDR; resulted in the recruitment of a new healthy ovarian follicle. The the majority of the cases developed new follicles either in response to a 2 ^nd GnRH treatment or spontaneously following CIDR withdrawal are ovulated resulting in 46.7% (schedule-A) or 57.1% (schedule-B) pregnancy rates. Thus, the incorporation of an intra vaginal progesterone-releasing device such as CIDR into ovsynch and timed artificial insemination program is recommended to increase the chances to treat the cases of cystic ovaries in dairy cows.