Comparison of Pregnancy Rates by Week from Stallion Exposure and Overall Pregnancy Rates in Pasture-Bred Mares Synchronized with CIDR and/or Prostaglandin F2alpha

There was no difference in pregnancy rates by week from stallion turnout or overall pregnancy rates between control mares and mares treated with controlled intravaginal drug release (CIDR) and prostaglandin F2α or prostaglandin alone. Eight-day exposure to CIDR devices usually resulted in mild vaginal discharge.

1. Introduction

There is a need to develop short, efficient, and affordable estrous synchronization protocols that align estrus and ovulation. Estrous synchronization in horses has been problematic because of the long duration of behavioral estrus and the variable time frame to ovulation. Many mare owners do not have access to a stallion for teasing to determine their mare’s receptivity for breeding. Access to a stallion or semen may be limited to specific days or dates. Therefore, many people in the equine industry routinely depend on estrus induction or synchronization as part of their breeding management.

Options for estrous synchronization in mares include the use of: oral synthetic progestagens (altrenogest), one or two injections of prostaglandin F2α, injectable progesterone and estradiol preparations, long-acting progesterone injection, and follicular ablation [1,2]. In addition, there are a few reports on the use of controlled intravaginal drug release (CIDR) devices impregnated with progesterone for manipulation of the reproductive cycle in mares [2]. In North America, the cattle device is the CIDR that contains 1.9 g of progesterone and has a release profile of 14 days. These devices are inexpensive and easy to insert. They are available in North America for use in cattle but are not labeled for use in horses. The application of these devices has been associated with mild vaginal discharge in cattle, but pregnancy rates were not affected by vaginal discharge [3]. The objective of this study was to compare pregnancy rates in mares synchronized for pasture breeding using no treatment, using a single injection of prostaglandin, or using an 8-day CIDR exposure with prostaglandin treatment on day 8.

2. Materials and Methods

The experimental protocol was approved by the University of Saskatchewan’s Institutional Animal Care and Use Committee. Fifty-one Belgian cross 2-yr-old mares were used for the study. Mares were maintained on pasture and individually identified by numbered freeze brands.

Mares were randomly assigned to treatment groups (n = 17 per group): group 1, untreated (control); group 2, prostaglandin F2α (Lutalyse [a], 5 mg, SC, day 0); group 3, CIDR device (inserted vaginally on day 0; removed and 5 mg of prostaglandin F2α, SC, injected on day 8. Day 0 was May 29th, 2003.

Ultrasound examinations were performed on the first examination day (day 0) using an Aloka 500 ultrasound machine [b] and a 5 mHz linear array probe [c]. Follicular size, the presence or absence of luteal tissue, intra-uterine fluid, endometrial edema (range 0 - 4) [d], and uterine and cervical tone were recorded. Mares were categorized as estrus, diestrus, or pubertal. The classifications were based on the following guidelines: estrus, follicle > 30 mm and soft uterine and cervical tone; diestrus, presence of distinct luteal tissue; pubertal, small ovaries, follicles <20 mm, and flaccid uterine tone.

After transrectal ultrasonographic examination, group 3 mares had their perineum cleansed, and a CIDR device was inserted into the mare’s vagina by an examiner wearing a new rectal sleeve lubricated with sterile methylcellulose gel. The plastic string was removed from the CIDR. The CIDR devices were removed on day 8, and vaginal discharge was assessed at the time of CIDR withdrawal: mild, mild creamy discharge detected only on the CIDR device; moderate, moderate amount of creamy, crusty discharge visible on the vulvar lips; severe, severe creamy, crusty discharge visible on the vulvar lips and resulting in scalding below the vulva.

On day 0, mares in groups 1 and 2 were turned out to pasture with Quarter horse stallions of proven fertility. Group 3 was turned out on day 8 after CIDR removal.

Pregnancy evaluation was performed by rectal palpation of mares in a portable chute in pastures. Examiners did not know the group number of the mares. Groups 1 and 2 were evaluated for pregnancy 64 days from stallion turn-out, and Group 3 mares were assessed 56 days from stallion turn-out. Duration of pregnancy was estimated by experienced examiners in intervals of 5 days (for example, ≥20, 25, 30, 35, etc. up to 65 days). The week in which mares conceived was determined by calculating backwards using the 5-day pregnancy duration intervals. Data were analyzed by week from stallion turn-out.

A significance level of p < 0.05 was used for all statistical tests. χ2 analysis was used to evaluate the frequency of mares in estrus, diestrus, or puberty at the time of the first examination. The frequency and degree of vaginal discharge in mares at the time of CIDR withdrawal on day 8 was recorded. Fischer’s exact test was used to compare group 3 with groups 1 and 2 in the proportion of mares with follicles >31 mm at the time of the initial examination and after the 8-day CIDR exposure. Differences in pregnancy rate between groups by week from stallion turn-out were compared using Kruskal Wallis one way analysis of variance (ANOVA) and χ2 analysis. A software program [e] was used for data analysis.

3. Results

Two mares were lost to follow-up (two mares in group 2). The CIDRs were retained by all group 3 mares (17/17, 100%). No mares had detectable free intra-uterine fluid at any of the examination times. Vaginal discharge was noted at CIDR removal and was found to be mild in 13 of 17 mares (76%), moderate in 4 of 17 mares (24%), and severe in 0 of 17 mares (0%). Six mares in group 3 were classified as estrus, and four of these mares had large follicles (50 mm, 50 mm, 45 mm, and 40 mm, respectively) at the time of CIDR insertion. These four mares had corpora lutea at CIDR withdrawal and had a confirmed pregnancies staged as 50, 35, <20, and 30 days, respectively. χ2 analysis did not show any differences (p > 0.05) between mare groups in the frequency of mares in estrus (group 1, 6 of 17; group 2, 9 of 17; group 3, 6 of 17), diestrus (group 1, 10 of 17; group 2, 7 of 17; group 3, 10 of 17), or pubertal (groups 1 - 3, 1 of 17 each) at the start of the study. There were no differences (p > 0.05) between groups in the proportion of mares with follicles >31 mm at day 0 (group 1, 7 of 17; group 2, 12 of 17; group 3, 9 of 17) or the total number of follicles >31 mm (group 1, 10 of 17; group 2, 12 of 17; group 3, 10 of 17). Additionally, there were no differences between the proportion of group 3 mares on day 8 with follicles >31 mm or the total number of follicles >31 mm (12 of 17) when compared with groups 1 - 3 at day 0. There were no differences (p = 0.7522) between groups in the pregnancy rate by week from stallion turn-out or in the frequency of pregnancy by week (p = 0.3290).

Figure 1 shows the distribution of pregnancies by week from stallion turn-out for each group. Overall pregnancy rates were 94% for group 1 (16 of 17), 100% for group 2 (15 of 15), and 88% for group 3 (15 of 17).

Figure 1. Comparison of pregnancy rates by week from stallion turn-out in untreated, single injection prostaglandin (PG), and 8-day CIDR- and PG-treated pasture-bred mares.

4. Discussion

The CIDR device is a low-cost, convenient method of delivering steady state levels of progesterone in cattle. They are easy to insert. The data show that the CIDR device had a high retention rate in mares. These devices may be used to ameliorate estrous behavior in mares, stimulate ovarian activity in transitional mares, and synchronize estrus [2]. There are preliminary reports on the use of these devices to synchronize estrus and terminate an estrus in mares [2,4].

An 8-day treatment period with the CIDR device resulted in some vaginal discharge in all treated mares. The discharge was characterized as mild in 13 of 17 mares. There is a report of a 12-day CIDR treatment where a slight vaginal discharge was noted at time of CIDR removal in 39 of 40 mares [5]. These findings suggest that CIDR devices may not be suitable for mares with poor perineal conformation that predisposition to vaginal contamination, cervical problems, or susceptibility to endometritis because of the potential for ascending infection. There is a similar intravaginal progesterone-releasing device that is reported to create less pressure and irritation of the vaginal tissue [6]. Vaginal mucous and associated vaginal irritation has also been reported with use of CIDRs in cattle. Its use has generally not been regarded as a factor that adversely affects fertility, because no cause and effect relationship can be made between higher vaginal mucous scores and lower pregnancy rates [3]. This observation and the observations in horses strongly support the use of proper sanitary conditions during CIDR application and removal to reduce the degree of vaginal contamination and subsequent vaginitis that may have a negative effect on pregnancy rates.

There were no pre-existing differences in the mare groups in the frequency of mares in estrus, diestrus, or pubertal. Five-day intervals were used to stage the duration of pregnancy, because, in the examiner’s experience, this was the degree of precision that could be obtained using this technique. The finding of no difference between groups in pregnancy rate by week after stallion turn-out indicates that neither prostaglandin F2α-treated mares or CIDR plus prostaglandin F2α-treated mares were synchronized significantly better than untreated mares. The lack of synchronization in the prostaglandin-treated mares may be associated with a failure to select mares for the presence of luteal tissue or with not using two injections of prostaglandin spaced 14 days apart. Better synchronization may have been seen if the selection criterion for this group had included mares with a diestrus status.

Four of the six estrus mares in group 3 with large follicles were found to have luteal tissue at CIDR withdrawal. Looking at the distribution of pregnancy duration of these four mares, it seems that the luteal tissue was not susceptible to prostaglandin in three of the mares, because they had a pregnancy duration of <35 days. This indicates that they were bred= 3 wk after stallion turn-out. The remaining mare began the CIDR treatment with a 50-mm follicle. This follicle may have ovulated immediately after CIDR insertion such that the luteal tissue was susceptible to prostaglandin. The pregnancy duration of 50 days suggests that she was bred in the first week after stallion turn-out. A 12 - 14-day CIDR insertion period may increase the proportion of mares responding to prostaglandin after CIDR removal. If a longer CIDR exposure period is used, more estrus mares that subsequently ovulate during the period of CIDR exposure would be expected to respond to prostaglandin, and thus, increase the overall number of mares synchronized.

There are reports that CIDRs are effective in promoting follicular development in transitional mares and in synchronizing estrus but not ovulation in mares during the breeding season [5]. These studies state that the addition of a deslorelin implant accelerated ovulation but did not increase ovulation synchrony because of the uncontrolled patterns of follicular development. We observed that the CIDR did not inhibit ovulation in 23.5% of estrus mares in group 3. Lubbecke et al. [5] reported that the use of CIDR for 12 days increased the proportion of mares with follicles >31 mm. Using an 8-day CIDR exposure, we were unable to detect a difference in the follicle numbers >31 mm or the frequency of mares with follicles >31 mm when comparing the data from day 0 of the same group 3 mares or day 0 of groups 1 and 2. It is possible that our findings may be explained by the different exposure period (8 versus 12 days), a breed effect (Hanoverian mares versus Belgian mares), or chance. Klug and Jöchle [2] reported that both estrus and ovulation could be synchronized if mares were exposed to CIDR for 12 days, given 10 mg of estradiol on day 1, prostaglandin on day 12, and treated with a deslorelin implant when a follicle >40 mm was detected.

In summary, a proportion of mares treated with CIDR during estrus will ovulate. Additionally, the vaginal discharge associated with an 8-day CIDR exposure is generally mild and does not influence pregnancy rates in pasture-bred mares. Vaginal discharge may be viewed as unsatisfactory for performance horse owners. Mares with poor conformation may not be suitable candidates for CIDR use, because they may be more susceptible to vaginal and uterine inflammation. An 8-day CIDR exposure did not promote the development of a greater proportion of follicles >31 mm in this population of young cycling mares. The lack of a difference in pregnancy rates between treatment groups indicated that none of the treatments significantly influenced fertility (i.e., pregnancy rates) compared with untreated control mares. Further investigation is required to identify treatments that create a more synchronous or earlier emergence of a follicular wave in a short timeframe and determine the type, dose, and duration of treatment with progesterone.

Thanks to the Alberta Agriculture Research Institute and the Equine Health Research Fund of the Western College of Veterinary Medicine for financial support.

Footnotes

1. Upjohn Pharmacia, Orangeville, Ontario, Canada L9W 3T3.
2. Aloka 500 ultrasound machine, Aloka Co LTD, 6-22-1 Mure, Mitaka-Shi, Tokyo 181 Japan.
3. Aloka Co LTD, 6-22-1 Mure, Mitaka-Shi, Tokyo 181 Japan.
4. Adams GP. Unpublished data, 1992.
5. Statistix 7, Analytical Software, PO Box 12185, Tallahassee, FL 32317-2185.