Optimizing Pregnancy Rates Using Frozen-Thawed Equine Semen

In an equine private practice, pregnancy rates using equine frozen-thawed semen seem significantly higher when inseminating mares with 600-800 million progressively motile sperm (PMS) compared with <600 and >800 million PMS.

1. Introduction

In an artificial insemination program using fresh semen, the minimum insemination dose necessary for maximum pregnancy rates has long been accepted to equal 500 million progressively motile spermatozoa (PMS) inseminated every 48 h while the mare is estrus [1]. More recently, however, it is suspected that this minimum dose is likely stallion-dependent. Furthermore, the site of insemination (uterine body versus uterine horn versus oviductal papilla versus oviduct) plays a role in the minimal number of spermatozoa required for acceptable pregnancy rates.

Many studies that examine the fertility of frozen-thawed semen have been published [2-4]. Few studies, however, have been published that examine the actual number of frozen-thawed motile spermatozoa that yield optimal pregnancy rates in the mare [5], and none have been published, to the author's knowledge, that use frozen semen from a large number of stallions, representing variable breeds and registries, in a private practice. This study examines the number of frozen-thawed progressively motile sperm that are necessary to optimize pregnancy rates when inseminating mares with cryopreserved semen.

2. Materials and Methods

Ninety mares were bred over 312 cycles with frozen-thawed semen from a total of 46 stallions. The breeds of stallions represented in the study included Thoroughbreds, American Saddlebreds, European Warmbloods, Quarter Horses, Paint Horses, Morgans, Arabians, and Welsh ponies. The stallions included in this study had at least one recorded pregnancy from insemination with their frozen-thawed semen.

Mares in estrus were examined daily by transrectal palpation and ultrasound examination [a]. When endometrial folds and a dominant follicle were readily detected by ultrasound examination, an ovulation induction agent (either an intramuscular injection of 1500 - 5000 i.u. human chorionic gonadotropin (hCG) [b] or deslorelin [c]) was administered. The experiment was designed to breed mares at 36 and 42 - 44 h after administration of the induction agent, with ovulation occurring in between inseminations; the intent, therefore, was to breed mares both pre- and post-ovulation. Mares were bred with an initial dose of frozen-thawed semen at 36 h after administration of the induction agent. If the first insemination occurred after ovulation, the mares were not re-inseminated at 44 h. Likewise, if only a single straw of semen was available for breeding, the mare was bred only at 36 h after administration of the ovulation induction agent. Finally, if the mare failed to ovulate by 44 h after induction, she was examined every 6 h and re-bred as soon as ovulation was confirmed by ultrasound examination.

Mares were inseminated using a rigid insemination pipette [d], passed transcervically into the uterine body or horn. Transrectal manipulation of the insemination pipette was not performed. The total volume of semen ranged from 0.5 to 5 ml per inseminate.

The total number of sperm inseminated into each mare, per cycle, ranged from <100 to >800 million PMS. If intraluminal fluid was detected by ultrasound either pre- or post-insemination, the mares were treated with either one or a combination of treatments - oxytocin [e] (10 i.u., IM), cloprostenol [f] (125 μg, IM), or intrauterine lavage with lactated Ringer solution [g]. Pregnancy was confirmed either by ultrasound examination 12 days or more after ovulation or by embryo recovery 7 - 8 days after ovulation.

The author, in all cases, subjectively estimated both total and progressive motility of the frozen-thawed spermatozoa. The percentage of motile sperm was estimated using a phase-contrast microscope [h] with a stage warmer set at 37°C. In some cases, the subjective analysis was confirmed by objective computer-assisted motility analysis.

The author used a χ2 analysis with ProcFreq from SAS 9.1 to analyze data for significance at p < 0.02.

3. Results

The pregnancy results with respect to the total number of sperm inseminated per cycle are depicted in Table 1 and Fig. 1.

Figure 1. χ2 analysis with ProcFreq of SAS 9.1 was used to analyze data for significance at p < 0.02. A is different than B (p < 0.2).

Overall, the per cycle pregnancy rate for mares bred over 312 cycles equaled 68%, with pregnancies recorded for 212/312 cycles. The pregnancy rates for mares inseminated with <200, 200 - 400, 400 - 600, 600 - 800, and >800 million PMS were 54% (12/22), 70.2% (66/94), 60% (30/50), 88.2% (60/68), and 56.4% (44/78), respectively. Pregnancy rates of mares bred with <200, 200 - 400, 400 - 600, and >800 million PMS were not statistically different (p > 0.02). Conversely, mares bred with 600-800 million PMS had significantly higher pregnancy rates than the mares bred with other doses (p < 0.02).

Although the intent of the experimental design was to breed mares both pre- and post-ovulation, of the 312 ovulatory cycles covered, mares were bred with a single post-ovulation dose on 34 cycles and had a pregnancy rate of 47% (16/34). Additionally, mares that were bred with a single pre-ovulatory dose on 44 cycles showed a pregnancy rate of 72% (32/44). The remainder of the 234 cycles was covered with both pre- and post-ovulation inseminations and resulted in a pregnancy rate of 70% (164/234).

4. Discussion

One of the disadvantages to breeding mares with frozen semen is that the fertility of frozen semen may be considerably lower than fresh semen, especially with respect to the age of the mare [6]. A number of recent studies have challenged this perception. Although foaling rates and per cycle pregnancy rates cannot be directly compared, the per cycle pregnancy rates reported in this study, regardless of sperm number, are encouraging with respect to the 2004 foaling rate of almost 64% recorded by the Jockey Club [i]. Others have reported similar per cycle and per season pregnancy rates with the use of frozen-thawed semen [2,3]. Samper et al. [3] reported a study in which 876 mares were bred with frozen-thawed semen from 106 stallions; the per cycle and per season pregnancy rates were 53.5% and 81.9%, respectively.

This study not only shows expected pregnancy rates for breeding mares in private practice with frozen-thawed semen but also the effect of motile sperm number in the inseminate on pregnancy rate. Pickett et al. [7] recommended a minimum insemination dose of 280 million PMS (800 million total sperm with at least 35% motility) when breeding with frozen semen to optimize pregnancy rates. A previous study performed by Leipold et al. [8] examined mares bred with frozen-thawed semen from five stallions; they compared pregnancy rates when breeding with 320 versus 800 million motile frozen-thawed sperm and found pregnancy rates to be 27% and 37%, respectively. These researchers concluded that frozen-thawed sperm doses should contain at least 320 million motile sperm.

Although the minimum insemination dose is suspected to be highly stallion-dependent, results from this study suggest that, in general, a dose closer to 600 - 800 million PMS will provide higher pregnancy rates. Squires et al. [5] reported similar high pregnancy rates in a field trial using frozen-thawed semen, whereby mares were bred with a minimum of 250 million PMS within 6 h after ovulation (83%) or at 24 and 40 h after hCG administration (86.6%). There were seven stallions used in the study; they were presumed to have unusually high fertility. This study presented far lower values after post-ovulation insemination; perhaps semen from certain stallions possesses intrinsic factors that make it more suitable for post-ovulation breeding.

It seems that acceptable pregnancy rates can be obtained with very low doses of fresh and frozen-thawed semen when inseminated closer to or within the oviduct. Morris et al. [9] showed a 67% pregnancy rate when breeding a total of 86 mares with a single insemination dose from two stallions with as few as 14 million fresh motile sperm placed hysteroscopically at the uterotubal junction ipsilateral to the dominant follicle. Deep horn insemination with as few as 5 million total sperm has yielded a 35% pregnancy rate. Lindsey et al. [10] showed a 37.5% pregnancy rate in mares that were bred with 15 million motile frozen-thawed sperm inseminated by hysteroscopy at the uterotubal junction. In the same experiment, if the frozen-thawed semen was subjected to flow-cytometric sorting, pregnancy rates dropped to 13.3%.

The positive pregnancy results obtained from low-dose inseminations, even with frozen-thawed semen, raise questions regarding not only the dosage of sperm required for minimum insemination that will optimize pregnancy rates but also the number of breeding doses obtained from a single ejaculate. If more breeding doses can be obtained per ejaculate, the cost incurred to the stallion owner will likely become lower.

Low-dose insemination techniques require additional time, training, staff support, and often, expensive equipment. The techniques seem essential when only very low doses of sperm are available. In this study, only a single veterinarian without specialized equipment was required for uterine body insemination - a procedure that yields efficient and effective results.

It is interesting to note, in this study, the significant decrease in pregnancy rates seen when mares are inseminated with > 800 million frozen-thawed PMS. There are several potential explanations to be considered. First, both the mares bred and semen used may have been from a less fertile population of both mares and stallions. Therefore, in attempt to compensate for unsuccessful breeding attempts, higher doses of sperm may have been used in subsequent breedings. Thus far, compensable defects, like those found in bull semen, have yet to be proven in the stallion. Second, the high number of sperm and the relative absence of seminal plasma may have caused a severe inflammatory reaction unable to be cleared by some mares. Third, perhaps if the sample size was larger and the breeding stock was selected based on its fertility, this trend would not be apparent.

Likewise, by limiting this study to examine only the effect of PMS number on pregnancy rate, many of the factors that contribute to equine pregnancy rates, including but not limited to, the age of the mare and stallion, specific sperm characteristics (especially those highly correlated with fertility), the reproductive status of the mare, the number of cycles per successful pregnancy, the interval (if any) between first and second inseminations, or post-insemination treatments, were not analyzed statistically. A more robust study, involving a larger population size, would result if these factors were considered.

Last, the European Warmblood stallion was over-represented in this study in that of semen from 46 stallions, 34 were Warmblood stallions. In Europe, many Warmblood registries select stallions for semen quality and potentially heritable traits to represent their breeding stock; hence, the pregnancy rates cited in this study may be higher than those seen in practices that use semen from breeds that select their breeding stallions based on show performance rather than semen quality.

Footnotes

1. Aloka Co. Ltd., Wallingford, CT 06492.
2. Chorulon; Intervet Inc., Millsboro, DE 19966.
3. Ovuplant; Fort Dodge Animal Health, Fort Dodge, IA 50501.
4. Veterinary Concepts, Spring Valley, WI 54767.
5. ProLabs, St Joseph, MO 64503.
6. Estrumate; Schering Plough Animal Health, Union, NJ 07083.
7. Lactated Ringer's Solution, Baxter Healthcare Co., Deerfield, IL 60015.
8. Olympus CH-II, Rocky Mountain Microscope Co., Fort Collins, CO 80524.
9. Cooney J. Personal communication.