The influence of mare numbers, ejaculation frequency and month on the fertility of Thoroughbred stallions

Reason for performing the study: Although considerable variation in per cycle pregnancy rates exists between Thoroughbred (TB) stallions, there is little information on factors that may influence this figure. Objective: To assess the influence of month, mare numbers and mating frequency on the fertility of TB stallions standing on studfarms in East Anglia, England. Methods: The daily breeding records of 31 TB stallions mating 3034 mares on 4851 occasions during the 2010 season were surveyed and related to first scan pregnancy rates. The influences of mare book size, month, number of matings per day and mating frequency or abstinence on per mating pregnancy rates were analysed. Results: The overall per mating pregnancy rate for all the stallions was 59.6%, but for individual stallions the figures ranged from 19.0% to 80.1%. The first mating occurred on 9 February and the last on 24 July and the per mating pregnancy rate per month was significantly reduced in June and July. The number of mares mated by individual stallions ranged from 15 to 161, giving a mean overall workload of 160 matings per 100 mares. The per mating pregnancy rate was not related to book size, the number of matings in the season or the mating frequency per day. However, some stallions showed differences in per mating pregnancy rate related to month or the number of ejaculations in the preceding 3 days. Conclusions: The majority of TB stallions are able to maintain good fertility despite large books of mares. However, 5 of the 31 stallions surveyed showed a per mating pregnancy rate of ≤50%. Potential relevance: This survey has identified wide differences between the per mating pregnancy rate in TB stallions. Identification of the factors involved through more comprehensive surveys would provide useful information for mare and stallion owners.     

Reproductive efficiency of Flatrace and National Hunt Thoroughbred mares and stallions in England

REASONS FOR PERFORMING STUDY: Previous surveys of reproductive efficiency in British Thoroughbreds included only mares and stallions standing on studfarms in and around Newmarket. The present study was widened to compare Flatrace (FR) (Group A) and National Hunt (NH) (Group B) mares and stallions on studfarms throughout England. OBJECTIVES: To assess the influences of mare type, status and age, and veterinary manipulations on reproductive efficiency parameters. To compare the inherent fertility of stallions, based on singleton and twin pregnancy rates and pregnancy loss rates, in Groups A and B Thoroughbred breeding stock. METHODS: Managers of 24 FR and 9 NH public studfarms were asked to complete a questionnaire for each mated oestrous cycle shown by 2321 Group A and 1052 Group B mares throughout the 2002 mating season. Parameters such as per cycle singleton and twin pregnancy rates, and pregnancy loss rates were noted, and the success of hormone treatments to induce oestrus and ovulation assessed. The number of matings per oestrus and per pregnancy were recorded, together with the incidence and effectiveness of uterine and other veterinary treatments. The inherent fertility of 84 Group A and 43 Group B stallions in the study, as measured by the singleton and twin early pregnancy rates and the pregnancy loss rates recorded in the mares they mated, was also estimated. RESULTS: Per cycle early pregnancy (Days 13-16) was 63.2% for Group A and 65.3% for Group B mares; and 10.3% and 13.1%, respectively, of those pregnancies were twins or triplets. Early, middle and late pregnancy loss rates were 7.2% vs. 8.0% (Days 15-42), 3.6% vs. 6.1% (Days 42-1st October) and 2.7% vs. 2.1% (October-foaling), respectively. Matings per oestrus and per early pregnancy were significantly higher in Group B vs. Group A mares. For stallions that mated > or = 30 mares, overall early pregnancy rates per cycle in mares mated ranged from 30-89% across the 2 groups. CONCLUSIONS: No major differences in reproductive efficiency were identified between FR and NH mares and stallions. Increasing mare age was the single biggest limiting factor to an otherwise high rate of fertility in well-managed English Thoroughbreds. POTENTIAL RELEVANCE: This study identified factors that influence reproductive efficiency in the Thoroughbred.     

Fertility at the first post partum estrous compared with fertility at the following estrous cycles in foaling mares and with fertility in nonfoaling mares

A retrospective study on the reproductive performance of 401 artificially inseminated trotter mares during six breeding seasons is presented. Mares, 279 post partum (PP) and 122 maiden and barren, or nonlactating (NL), were inseminated with fresh semen obtained from four fertile stallions of the same breed. Pregnancy rate (PR) of mares inseminated at the foal heat (182/253, 71.9%) was lower, but not significantly different, than the PR (22/26, 84.6%) of mares inseminated for the first time at the second post partum cycle and similar to the PR at the first and second cycle of NL mares (95/112, 77.8% and 25/33, 75.7%, respectively). PR of mares inseminated at the foal heat was higher, but nonsignificantly different, from PR of the post partum mares not pregnant after artificial insemination (AI) at foal heat and inseminated again at the following estrous cycle. The PR after AI at the foal heat was significantly higher than the PR when the AI was performed at the third or later cycle in NL mares (71.9% vs 22.2%, P<0.01). The estrus cycle/pregnancy ratio for the PP mares inseminated, for the first time at the foal heat or at the second heat and for NL mares was, respectively, 1.4, 1.2, 1.3 at first cycle and 1.4, 1.3 and 1.4 at the end of the season or when mares left the stud. The proportion of open mares at the end of the season or when leaving the stud was 7.1% (18/253), 3.8% (1/26) and 4.1% (5/122) for PP mares first inseminated at the first or second post partum cycle and for NL mares, respectively; the total rate of open mares was 6% (24/401). The foaling rate (FR) following conception at the foal heat (72.1%) was not statistically different from the FR following conception at any other cycle (50–100%). Based on the absence of significant differences in fertility at the first post partum estrus cycle versus any other estrus cycle, we conclude that breeding at the foal heat should be advisable.     

Key Factors Affecting Reproductive Success of Thoroughbred Mares and Stallions on a Commercial Stud Farm

To evaluate factors contributing to fertility of thoroughbred mares, data from 3743 oestrous periods of 2385 mares were collected on a large thoroughbred farm in Ireland. Fourteen stallions (mean age 8.3 years; range 4–15 years) had bred 2385 mares (mean age 9.4 years; range 3–24 years). Maiden mares accounted for 12%, mares with a foal at foot for 64%, and barren, slipped or rested mares for 24% of the total. The mean pregnancy rate per cycle was 67.8% (68.6% in year 1 and 66.9% in year 2). Backward stepwise multivariable logistic regression analysis was utilized to develop two models to evaluate mare factors, including mare age, reproductive status, month of foaling, dystocia, month of cover, foal heat, cycle number, treatments, walk‐in status and stallion factors including stallion identity, stallion age, shuttle status, time elapsed between covers and high stallion usage on the per cycle pregnancy rate and pregnancy loss. Old age (p < 0.001) and cover within 20 days post‐partum (p < 0.003) were associated with lowered pregnancy rates. High mare age (p < 0.05) and barren, slipped or rested reproductive status (p = 0.05) increased the likelihood of pregnancy loss. Uterine inflammation or infection, if appropriately treated, did not affect fertility. Only high usage of stallions (used more than 21 times in previous week) was associated with lowered (p = 0.009) pregnancy rates. However, shuttle stallions were more likely to have increased (p = 0.035) pregnancy survival, perhaps reflecting a bias in stallion selection. In conclusion, mare age exerted the greatest influence on fertility; nonetheless, thoroughbreds can be effectively managed to achieve high reproductive performance in a commercial setting.     

Management Strategies Aiming to Improve Horse Welfare Reduce Embryonic Death Rates in Mares

The objective of this retrospective study was to evaluate the effect of management strategies aiming to improve animal well‐being on pregnancy and embryonic death (ED) rates. Breeding records of a cohort of 1206 Thoroughbred mares brought to a stallion station facility, to be bred with the stallions housed there, were evaluated during ten breeding seasons. Mares were blocked according to management strategies in two groups: Stress and Relax. Strategies used to improve animal well‐being (Relax group) were as follows: stopping the teasing routine, reducing or eliminating stall confinement, reducing the number of mares per group and maintaining herd stability during the breeding season. In barren mares, the pregnancy rate was higher in the Relax group (91.8%) when compared to the observed in Stress group (84.7%). However, no difference in pregnancy rates were observed (Stress = 85.2% vs. Relax = 86.2) in foaling mares. ED rate was higher in barren and foaling mares of the Stress group mares (25.5% and 26.8%, respectively) compared with the Relax group (16.1% and 14.7%, respectively). No significant differences were observed on foal heat pregnancy rate between groups; yet, the embryo loss on foal heat was significant reduced in Relax mares (Relax = 8.7% vs Stress = 24.5%). In conclusion, management strategies aimed to reduce social stress can reduce early pregnancy losses and the average cycles per pregnancy, improving reproductive performance in mares.     

Insemination Results with Slow-Cooled Stallion Semen Stored for approximately 40 Hours

Heiskanen, M.-L., M. Huhtinen, A. Pirhonen and P. H. Mäenpää: Insemination results with slow-cooled stallion semen stored for approximately 40 hours. Acta vet. scand. 1994,35,257-262.– Semen from 3 stallions was extended using 2 methods (Kenney extender and a modified Kenney extender), slowly cooled, and stored for 41 ± 6 (s.d.) h before insemination. An insemination dose (40 ml) contained 1.5-2 billion spermatozoa. In the experiment, 26 mares were inseminated in 30 cycles. The pregnancy rate per cycle obtained with sperm stored in the Kenney extender was 87% (n=15). When the semen was extended with the modified extender, centrifuged and stored, the pregnancy rate was 60% (n=15). Inseminations were done every other day until ovulation was detected. If a mare ovulated more than 24 h after the last insemination, she was inseminated also after ovulation. The single-cycle pregnancy rate was 58% when the mares were inseminated only before ovulation (n=19) but the rate was 100% when the inseminations were done both before and after ovulation (n=9) or only after ovulation (n=2). The difference in pregnancy rates was significant (p<0.05), indicating that postovula-tory inseminations probably serve to ensure the pregnancies. The extending and handling methods used in this study resulted in a combined pregnancy rate of 73%, and appear thus to be useful for storing stallion semen for approximately 2 days.     

Reproductive performance measures among Thoroughbred mares in central Kentucky,during the 2004 mating season

Reason for performing study: To improve efficiency at the farm level, a better understanding of how farm management factors impact reproductive performance is important. Objective: To assess reproductive efficiency and effectiveness among Thoroughbred mares in central Kentucky. Methods: A cohort of 1011 mares on 13 farms in central Kentucky was followed during the 2004 mating and 2005 foaling season. Information on farm level practices was collected via interviews with farm managers. Reproductive records were collected for each mare mated to obtain information on mare characteristics. The influence of mare age and status (maiden, foaling, barren) on Days 15 and 40 post mating pregnancy rates, foaling rates and total effective length of the mating season were assessed. The influence of stallion book size on reproductive performance measures was also examined. Results: Per season pregnancy rates on Days 15 and 40 post mating and live foal rate were 92.1, 89.3 and 78.3%, respectively. Per cycle rates for the same time periods were 64.0, 58.3 and 50.8%. There were no significant associations between stallion book size and reproductive performance outcomes. The mean s.d. interval from the beginning of the mating season to the last mating of the mare was 36.5 ± 26.1 days. Conclusions: Mare age had a significant impact on efficiency of becoming pregnant, maintaining pregnancy and producing a live foal. Overall, fertility did not decrease among stallions with the largest book sizes. Total interval length of the mating season can be reduced if managers ensure maiden and barren mares are mated at the beginning of the season and foaling mares are mated at the earliest oestrus after acceptable uterine involution has been achieved. Potential relevance: Measures identified in the study can be used by owners, farm managers and veterinarians to improve mare reproductive performance and identify parameters to assist with the implementation of effective culling practices.     

Reproductive Performance of Thoroughbred Mares in Sweden

During 1997–2001, a total of 430 Thoroughbred mares were mated by one of the two Thoroughbred stallions on a well‐managed stud farm in the central part of Sweden. On arrival, a thorough gynaecological examination of all mares was performed. An early pregnancy examination was performed on days 14 and 15 after ovulation. The overall conception rates for the two stallions were 92.2 and 88.8%, and the corresponding live foal rates were 82.6 and 75.2%, respectively. The mean number of served oestrous cycles was 1.60 per mare per conception and 1.86 per live foal. The first and second cycle conception rates are reported for different age groups, mare categories and month of mating. The age of the mares had a significant influence on the live foal rate, being lower for mares >13 years of age. Resorption and abortion occurred in higher percentages among mares >8 years of age. The highest embryonic death occurred among mares with a history of being barren, or with having resorbed or aborted foetuses during the previous season. The month of mating had no significant influence on the first cycle conception rate or the second cycle conception rate. The frequency of twinning was 10.5% and the results of manual crushing of one of the conceptuses was successful in 88.9% of all cases, according to the ultrasound scanning 2 days later.     

The effect of liposomes composed of phosphatidylserine and cholesterol on fertility rates using frozen thawed equine spermatozoa

The objective of this study was to determine if the addition of liposomes composed of phosphatidylserine (PS) and cholesterol (CH) to equine sperm would improve pregnancy rates after sperm were cryopreserved. Ejaculates from four stallions, collected every other day during May and June were split, treated with PSCH liposomes or HBS (Hepes Buffered Saline) and cryopreserved. Fifty-two mares were bred over eighty estrous cycles with the frozen-thawed semen. The one cycle pregnancy rates of the mares inseminated with semen treated with liposomes (45%) were similar to mares inseminated with control semen (48%; p>0.05). Thus, at least at the levels used, PSCH liposomes added to equine sperm did not improve pregnancy rates of mares inseminated with cryopreserved semen.     

Male, female and management risk factors for non-return to service in Dutch mares

The "effect" of stallion, mare and management-related factors on the odds of pregnancy per cycle in the horse were identified and quantified from the breeding records of Dutch Warmblood (n=4491), Friesian (n=1467) and Shetland-pony mares (n=3267) mated either naturally or by artificial insemination to one of the 88 stallions between 1992 and 1996. A mare was considered to be pregnant when she did not return to oestrous within 28 days of the last insemination. For Dutch Warmblood horses, the percentage of mares that did not return for service within 28 days (NR28) varied between studfarms and ranged from 61 to 82%. The NR28 for mares inseminated with fresh semen ranged from 67 to 74% and for mares inseminated with frozen/thawed semen this percentage was 59. Mares served at a second cycle had lower odds not to return than mares served at the third or subsequent cycle (OR=0.84). For Friesian horses, the NR28 for young mares was higher than that for older mares. Mares served before 1 May in any year had lower odds of non-return than mares served after 1 July (OR=0.69). The NR28 of mares inseminated once per cycle was 6% lower than that of mares inseminated three times or more per cycle. For Shetland ponies, the NR28 also varied between studfarms and ranged from 62 to 78%. Stallions < or =3 years old had lower odds of non-return compared to older stallion (> or =11) (OR=0.57). Mares served before 1 July had lower odds of non-return. Other significant factors for this breed were age of the mare, cycle number and insemination frequency. Stallion factors accounted for 5.9, 2.0 and 14.7% of the variation in the NR28 for Dutch Warmblood, Friesian horses and the Shetland ponies, respectively.     

Retrospective study on the incidence of postinsemination uterine fluid in mares inseminated with frozen/thawed semen

Uterine fluid accumulation has been reported after insemination or natural breeding of mares. This retrospective study examined the factors affecting the incidence of uterine fluid after insemination of frozen semen. Specifically, this study determined the association between mare age, reproductive status, fluid accumulation, and pregnancy rates in mares. Records were available from 283 warmblood mares throughout 496 cycles. Mares were divided into maiden, foaling, and barren and age groups of 3 to 9, 10 to 16, and more than 16 years. Mares were inseminated only once with frozen semen within 4 to 8 hours before or after ovulation. Ultrasound examinations were performed 12 to 18 hours after insemination. A depth of at least 20 mm of fluid was considered significant. Mares with less than 20 mm were treated with oxytocin, and those with more than 20mm of fluid were given oxytocin and uterine lavage. Pregnancy determination was performed at 14 to 16 and 30 to 50 days after ovulation. Fluid level of more than 20 mm was recorded in 25% of the cycles. Barren mares and aged mares (10-16 and > 16 years) had a higher incidence of uterine fluid accumulations. Per-cycle pregnancy rate was lower (45%) in mares with uterine fluid than in mares without uterine fluid (51%). This difference was primarily due to the reduction in fertility of mares who were older than 16 years and retained fluid after insemination. Apparently, oxytocin and lavage treatments provided acceptable fertility in the other groups of mares that had uterine fluid.

Introduction

Use of equine frozen semen is accepted by the majority of horse registries. According to several field studies,[1, 2, 3, 4 and 5] insemination of frozen semen has resulted in acceptable pregnancy rates. Postbreeding fluid accumulation is a physiologic inflammation that clears the uterus of foreign material such as excess spermatozoa, seminal plasma, bacteria, and extenders. [6, 7, 8, 9 and 10] Uterine fluid can be easily diagnosed with ultrasonography. [10, 11 and 12] Persistent postbreeding uterine fluid has been associated with a decrease in fertility after natural mating or artificial insemination (AI) of fresh semen. [11, 12 and 13] Predisposing factors to persistent fluid accumulations are reduced myometrial contractions, poor lymphatic drainage, large overstretched uterus, and cervical incompetence. [7, 14 and 15] Normal mares are able to expel uterine fluid quickly after inseminations, whereas susceptible mares accumulate fluid in their uterine lumen for more than 12 hours after breeding or insemination. [10]It is commonly stated that insemination with frozen semen leads to greater post-AI fluid accumulation than insemination with fresh or cooled semen or after natural mating. Apparently, there is only 1 controlled study on this comparison.[7] The authors reported that infusion of frozen semen resulted in a greater inflammatory response than natural breeding. In a field study, [16] 16% of mares naturally mated had persistent postbreeding fluid accumulations compared with a 30% rate reported for mares inseminated with frozen semen. [1 and 2] More recently, Watson et al. [17] reported a postbreeding fluid accumulation rate of 16%, which is identical to that reported for natural mating. [16] It is difficult to compare studies because details of mare selection and insemination or breeding frequencies are not always reported. Obviously, a higher proportion of barren and aged mares in a study would increase the incidence of postbreeding fluid accumulation. [1 and 2]The study presented herein was a retrospective study designed to determine the incidence of postbreeding fluid accumulation in a large number of mares inseminated with frozen semen. Associations were determined between mare age, reproductive status and fluid accumulation, and pregnancy rate in mares with and without uterine fluid accumulation.

Materials and methods

Mares

Records were available from 283 warmblood mares inseminated with frozen semen at the Cristella Veterinary Clinic in Italy during 1998 to 2001. Mares ranging in age from 3 to 20 years were inseminated with semen that was frozen in 10 centers and was from 34 stallions. The broodmare population was subdivided into 3 reproductive groups: 89 maiden mares (mean age, 7.2 years), 106 foaling mares (mean age, 9.4 years), and 87 barren mares (mean age, 11.9 years). Maiden mares older than 7 years were selected with biopsy scores of 1 or 2 only. Barren mares were open for no more than 2 consecutive seasons and had negative cytology and bacteriology scores. Age groups were divided as follows: 3 to 9 years (n = 132), 10 to 16 years (n = 137) and older than 16 years (n = 14). Data from 496 cycles were used. Distribution of the estrous cycles was 172, 157, and 167 in the maiden, foaling, and barren groups, respectively; and 224, 244, and 28 in the youngest, intermediate, and oldest groups, respectively.

Mare reproductive management and artificial insemination protocol

During estrus, all mares underwent a daily ultrasound examination with a 5-mHz transrectal probe (SA 600 Vet; Medison Inc., Seoul, South Korea) until 1 or more 35-mm ovarian follicles were detected. Ovulation was then induced by the intravenous administration of 2000 IU of human chorionic gonadotropin (hCG). Ultrasound examination was performed 12 hours after hCG treatment and then every 4 to 8 hours until ovulation occurred. Mares were inseminated only once within a period of 4 to 8 hours before or after ovulation. The semen used was thawed according to the distribution center's instructions and had the following minimum post-thaw quality requirements: not less than 200 × 10⁶ progressively motile spermatozoa per dose and a minimum of 30% progressive spermatozoal motility. Foaling mares were not inseminated at their first postpartum (“foal heat”) estrous period, because pregnancy rates are recognized to be lower than during the subsequent estrous periods.[18] During the first postpartum estrus, ovarian ultrasound scan examinations were performed every 2 to 3 days until an ovulation was detected. A prostaglandin F₂α injection was given 5 days later to short-cycle the mare.

Postinsemination monitoring

An ultrasound examination of the reproductive tract was performed 12 to 18 hours after insemination to detect any intrauterine fluid accumulation. The presence and depth of intrauterine fluid was recorded. Twenty millimeters or more of grade II or III intrauterine fluid[19] was recorded as a significant amount of fluid. Mares with less than 20 mm of fluid were treated with an intravenous injection of 20 IU oxytocin. For mares with more than 20 mm of fluid, oxytocin was administered, and the uterus was flushed daily with buffered saline solution: 1-L aliquots were infused and recovered until the recovered fluid was clear. In these mares, oxytocin treatment was repeated up to 3 times daily. Post insemination treatments were performed for no more than 4 days after ovulation had occurred.Pregnancy diagnosis was performed with ultrasound at 14 to 16 days after ovulation. Scans were then repeated at 30 and 50 days of gestation to confirm the presence in the uterus of an apparently healthy developing conceptus.

Statistical analysis

χ² Analysis was used to determine the effect of reproductive status and age on the incidence of fluid accumulation. In addition, the influence of persistent uterine fluid accumulation on pregnancy rates per cycle was determined for each reproductive class and age by using χ² analysis.

Results

The per-cycle pregnancy rate at 14-16 days after ovulation was 49.3% (245/496 cycles). By the end of the season, 245 of 283 mares (86.5%) were confirmed pregnant. Fluid level of at least 20 mm (grade II or III) was recorded in 126 of the 496 cycles (25.4%). Barren mares had a higher (P < .05) incidence of postbreeding fluid accumulation (64/167; 38.3%) than maiden (34/172; 19.7%) and foaling (28/157, 17.8%; Table 1) mares. The incidence of fluid accumulation was also higher in mares older than 16 years (19/28; 67.8%) than those aged 10 to 16 years (69/244; 28.2%) and 3 to 9 years (38/224; 17%). The incidence of uterine fluid was also higher (P < .05) for mares aged 10 to 16 years than those aged 3 to 9 years (Table 2). Overall, the per-cycle pregnancy rate was lower (P < .05) for mares with post-AI fluid accumulations than for those with no uterine fluid or only a small quantity of fluid (57/126, 41.9% vs 188/360, 56.2%). Pregnancy rates were similar (P > .05) for mares with or without uterine fluid when comparisons were made within maiden and barren mare groups. However, more foaling mares became pregnant when no fluid was detected after insemination. Pregnancy rate for this group (68.1%) was higher than that for maiden (44.2%) and barren (44.6%) mares (Table 3). Older mares with uterine fluid accumulations had a lower per-cycle pregnancy rate (36.8%) than mares in the same group but without fluid. Surprisingly, if no fluid was detected, the highest pregnancy rates were in mares older than 16 years ( Table 4).     

Freezability and Fertility Results with Uncentrifuged Stallion Semen

The first (1 to 3) sperm-rich fractions of the ejaculate were collected from 4 stallions using an open-ended vagina. The volume of the collected fractions was 12 ± 8 ml with a density of 475 ± 200 million spermatozoa/ml. Before freezing, the semen was diluted with a skim-milk based extender 1:1 to 1: 8 (volume of semen: volume of extender), depending on the initial sperm concentration to achieve a final concentration of 100 million/ml. The total number of spermatozoa in an insemination dose ranged from 0.7 to 1 billion spermatozoa. Within 12 h after ovulation, 48 mares were inseminated in 70 cycles. The total single-cycle pregnancy rate at day 21 was 24%, but varied from 10% to 33% per cycle among the stallions.     

Trypan Blue/Giemsa Staining to Assess Sperm Membrane Integrity in Salernitano Stallions and its Relationship to Pregnancy Rates

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty‐one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty‐five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June–July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June–July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut‐off value of 48% LSIA. Trypan blue‐Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor‐quality ejaculates.     

Capsule types of Klebsiella pneumoniae isolated from the genital tract of mares with metritis, extra-genital sites of healthy mares and the genital tract of stallions

A survey of K. pneumoniae was performed on cervical swabs, feces and nasal swabs of mares and on samples from the genital tract of stallions from 1980 to 1986 in south-western Hokkaido, Japan. K1 was the predominant type (79 of 88, 89.8%) in the metritis cases due to K. pneumoniae in mares of racing breeds. The same type was isolated from semen and swabs of the fossa glandis of 6 of 20 (30.0%) of the stallions of racing breeds. Heavily encapsulated and less heavily encapsulated K1 strains were isolated from the stallions. Mares bred to stallions carrying heavily encapsulated strains developed metritis, while those bred to stallions carrying less heavily encapsulated strains did not. K39 was isolated from cervical swabs solely from metritis-infected mares of draft breeds and not from any mares of the racing breeds examined. Untypable strains were isolated from cervical swabs in 7 of 88 (8.0%) metritis cases of mares of racing breeds and from semen in 7 of 19 (36.8%) stallions of racing breeds and they were predominant in feces (19 of 21, 90.5%) and nasal swabs (3 of 4, 75.0%) of healthy mares of racing breeds.     

Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction

REASONS FOR PERFORMING STUDY: To compensate for the wide variation in the freezability of stallion spermatozoa, it has become common veterinary practice to carry out repeated ultrasonography of the ovaries of oestrous mares in order to be able to inseminate them within 6-12 h of ovulation with a minimum of 300-500 x 10(6) frozen-thawed spermatozoa. Furthermore, in order to achieve satisfactory fertility, this requirement for relatively high numbers of spermatozoa currently limits our ability to exploit recently available artificial breeding technologies, such as sex-sorted semen, for which only 5-20 x 10(6) spermatozoa are available for insemination. OBJECTIVES: This study was designed to evaluate and compare the efficacy of hysteroscopic vs. conventional insemination when low numbers of spermatozoa are used at a single fixed time after administration of an ovulation-inducing agent. METHODS: In the present study, pregnancy rates were compared in 86 mares inseminated once only with low numbers of frozen-thawed spermatozoa (3-14 x 10(6)) at 32 h after treatment with human chorionic gonadotrophin (hCG), either conventionally into the body of the uterus or hysteroscopically by depositing a small volume of the inseminate directly onto the uterotubal papilla ipsilateral to the ovary containing the pre-ovulatory follicle. RESULTS: Pregnancy rates were similarly high in mares inseminated conventionally or hysteroscopically with 14 x 10(6) motile frozen-thawed spermatozoa (67% vs. 64%). However, when the insemination dose was reduced to 3 x 10(6) spermatozoa, the pregnancy rate was significantly higher in the mares inseminated hysteroscopically onto the uterotubal junction compared to those inseminated into the uterine body (47 vs. 15%, P < 0.05). CONCLUSIONS: When inseminating mares with <10 x 10(6) frozen-thawed stallion spermatozoa, hysteroscopic uterotubal junction deposition of the inseminate is the preferred method. POTENTIAL CLINICAL RELEVANCE: Satisfactory pregnancy rates are achievable after insemination of mares with frozen-thawed semen from fertile stallions 32 h after administration of human chorionic gonadotrophin (Chorulon). Furthermore, these results were obtained when mares were inseminated with 14 x 10(6) progressively motile frozen-thawed spermatozoa from 2 stallions of proven fertility.     

Reproductive performance of Thoroughbred mares on six commercial stud farms

The records of 1630 mare years from 6 Thoroughbred stud farms in south eastern Australia were analysed for the years 1981 to 1986. Overall pregnancy and foaling rates were 83.9% and 69.3%, respectively. When calculated per served oestrous cycle, pregnancy and foaling rates were 54.7% and 43.1%, respectively. Pregnancy and foaling rates were higher (P < 0.001) for mares 3 to 10 years of age than for older mares. There was no difference in the pregnancy rates of maiden, barren and foaling mares. The foaling rate was significantly higher (P < 0.001) in mares that became pregnant during the first served oestrous cycle (77.8%) than in mares that needed two served oestrous cycles to become pregnant (65.4%). Of all diagnosed pregnancies, 19.5% were not completed. Pregnancy loss was lower (P < 0.05) in maiden (12.4%) than in barren (19.7%) or foaling (20.9%) mares. Twins were diagnosed in 7.8% of all pregnancies. If one conceptus was lost without external interference, 84.1% of pregnancies went to term. If one conceptus was manually crushed, 55.9% of pregnancies were maintained. If prostaglandin was used to terminate twin pregnancies, 60% of mares so treated produced foals the following year.     

Studies on Motility and Fertility of Cooled Stallion Spermatozoa

This study on extended, cooled stallion spermatozoa aimed to compare the ability of three extenders to maintain sperm motility during 24 h of preservation, and to describe pregnancy and foaling rates after artificial insemination (AI) of stallion spermatozoa stored and transported in the extender chosen from the in vitro study. After 6 and 24 h of preservation, motility, both subjective and evaluated by the motility analyzer (total, progressive and rapid), was lower in non-fat, dried skim milk-glucose than in both other extenders: dried skim milk-glucose added to 2% centrifuged egg yolk, and ultra high temperature treated skim milk-sugar-saline solution added to 2% centrifuged egg yolk (INRA82-Y). Rapid spermatozoa and sperm velocity parameters, after 24 h, were significantly higher in INRA82-Y. In the fertility trial, semen collected from three Maremmano stallions, diluted in INRA82-Y, and transported in a refrigerated Styrofoam box, was used to inseminate 56 mares of the same breed. Pregnancy rates after the first cycle and per breeding season were significantly higher for the 31 mares inseminated in three AI centres (54.8 and 80.6%, respectively) than for the 25 mares inseminated at the breeder's facilities (28.0 and 52.0%). Foaling rates were not significantly different between the AI centres mares (54.8%) and the other mares (44.0%). In conclusion, INRA82-Y yielded satisfactory pregnancy and foaling rates, especially when employed in the more controlled situation of an AI centre, and can therefore be included among those available for cooled stallion semen preservation.     

Successful Timing of Ovulation Using Deslorelin (Ovuplant®) is Labour-saving in Mares Aimed for Single AI with Frozen Semen

To minimize the number of matings/inseminations, controlled ovulation has been practised since a long time ago. A potent short-term implant, releasing the GnRH analogue deslorelin (Ovuplant((R))) has been used in Australia and North America for several years for hastening the ovulation time in mares, but the product is not registered on the European market. This study was aimed to investigate: (1) ovulation time in mares implanted with Ovuplant when the largest follicle was 42 mm or more in size, (2) repeatability of ovulation time in successive oestruses when treated with Ovuplant, (3) pregnancy rate after single insemination with frozen-thawed semen near ovulation. This study included 11 mares, and altogether 17 timed ovulations. Follicular growth and ovulation were determined by palpation per rectum and by ultrasonography in the morning (at 7:00 hours) every second day until observation of a follicle of at least 42 mm in diameter. Then the mares were re-examined in the afternoon (at 19:00 hours), and an Ovuplant was inserted in the mucosa of the vulva. For detection of ovulation, the mares were palpated and ultrasounded repeatedly from 36-42 h after the insert. The mares were inseminated with frozen-thawed semen once at ovulation. All mares ovulated at 36-48 h after treatment and 94% at 38-42 h after treatment. The six mares that were treated at two oestruses ovulated at 39.9 and 39.7 h, respectively. Five of 11 mares (45.4%), inseminated with frozen-thawed semen at the first oestrous cycle were pregnant day 14-16 after ovulation. Using this protocol, there is no need of palpation/ultrasonography during night hours, and examination at 36 and 41 h after implantation might be enough for estimation of ovulation time.     

Superovulation in cycling mares using equine follicle stimulating hormone (eFSH)

Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates. Other potential clinical applications include improving pregnancy rates from frozen semen, treatment of subfertility in stallions and mares, and induction of ovulation in transitional mares. The objective of this study was to evaluate the efficacy of purified equine follicle stimulating hormone (eFSH; Bioniche Animal Health USA, Inc., Athens, GA) in inducing superovulation in cycling mares. In the first experiment, 49 normal, cycling mares were used in a study at Colorado State University. Mares were assigned to 1 of 3 groups: group 1, controls (n = 29) and groups 2 and 3, eFSH-treated (n = 10/group). Treated mares were administered 25 mg of eFSH twice daily beginning 5 or 6 days after ovulation (group 2). Mares received 250 (of cloprostenol on the second day of eFSH treatment. Administration of eFSH continued until the majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting on day 5 or 6. The treatment regimen was identical to that of group 2. Mares in all 3 groups were bred with semen from 1 of 4 stallions. Pregnancy status was determined at 14 to 16 days after ovulation.In experiment 2, 16 light-horse mares were used during the physiologic breeding season in Brazil. On the first cycle, mares served as controls, and on the second cycle, mares were administered 12 mg of eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time human chorionic gonadotropin (hCG) was administered. Mares were inseminated on both cycles, and embryo collection attempts were performed 7 or 8 days after ovulation.Mares treated with 25 mg of eFSH developed a greater number of follicles (35 mm) and ovulated a greater number of follicles than control mares. However, the number of pregnancies obtained per mare was not different between control mares and those receiving 25 mg of eFSH twice daily. Mares treated with 12 mg of eFSH and administered either hCG or deslorelin also developed more follicles than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles than control mares, whereas the number of ovulations from mares receiving eFSH followed by deslorelin was similar to that of control mares. Pregnancy rate for mares induced to ovulate with hCG was higher than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of the controls. Overall, 80% of mares administered eFSH had multiple ovulations compared with 10.3% of the control mares.In experiment 2, the number of large follicles was greater in the eFSH-treated cycle than the previous untreated cycle. In addition, the number of ovulations during the cycle in which mares were treated with eFSH was greater (3.6) than for the control cycle (1.0). The average number of embryos recovered per mare for the eFSH cycle (1.9 ± 0.3) was greater than the embryo recovery rate for the control cycle (0.5 ± 0.3).In summary, the highest ovulation and the highest pregnancy and embryo recovery rates were obtained after administration of 12 mg of eFSH twice daily followed by 2500 IU of hCG. Superovulation with eFSH increased pregnancy rate and embryo recovery rate and, thus, the efficiency of the embryo transfer program.

Introduction

Induction of multiple ovulations or superovulation has been an elusive goal in the mare. Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates.[1 and 2] Superovulation also has been suggested as a critical requirement for other types of assisted reproductive technology in the horse, including oocyte transfer and gamete intrafallopian transfer. [2 and 3] Unfortunately, techniques used successfully to superovulate ruminants, such as administration of porcine follicle stimulating hormone and equine chorionic gonadotropin have little effect in the mare. [4 and 5]The most consistent therapy used to induce multiple ovulations in mares has been administration of purified equine pituitary gonadotropins. Equine pituitary extract (EPE) is a purified gonadotropin preparation containing approximately 6% to 10% LH and 2% to 4% FSH.[6] EPE has been used for many years to induce multiple ovulations in mares [7, 8 and 9] and increase the embryo recovery rate from embryo transfer donor mares. [10] Recently, a highly purified equine FSH product has become available commercially.The objectives of this study were to evaluate the efficacy of purified eFSH in inducing superovulation in cycling mares and to determine the relationship between ovulation rate and pregnancy rate or embryo collection rate in superovulated mares.

Materials and methods

Experiment 1

Forty-nine normally cycling mares, ranging in age from 3 to 12 years, were used in a study at Colorado State University. Group 1 (control) mares (n = 29) were examined daily when in estrus by transrectal ultrasonography. Mares were administered an implant containing 2.1 mg deslorelin (Ovuplant, Ft. Dodge Animal Health, Ft. Dodge, IA) subcutaneously in the vulva when a follicle 35 mm in diameter was detected. Mares were bred with frozen semen (800 million spermatozoa; minimum of 30% progressive motility) from 1 of 4 stallions 33 and 48 hours after deslorelin administration. The deslorelin implants were removed after detection of ovulation.[11] Pregnancy status was determined at 14 and 16 days after ovulation.Group 2 mares (n = 10) were administered 25 mg of eFSH (Bioniche Animal Health USA, Inc., Athens, GA) intramuscularly twice daily beginning 5 or 6 days after ovulation was detected. Mares received 250 g cloprostenol (Estrumate, Schering-Plough Animal Health, Omaha, NE) intramuscularly on the second day of eFSH treatment. Administration of eFSH continued until a majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Mares were subsequently bred with the same frozen semen used for control mares, and pregnancy examinations were performed as described above.Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting 5 or 6 days after ovulation and were administered 250 μg cloprostenol on the second day of treatment. Mares were randomly selected to receive either a deslorelin implant (n = 5) or 2500 IU of human chorionic gonadotropin (hCG) intravenously (n = 5) to induce ovulation when a majority of follicles reached a diameter of 35 mm. Mares were bred with frozen semen and examined for pregnancy as described above.

Experiment 2

Sixteen cycling light-horse mares were used during the physiologic breeding season in Brazil. Reproductive activity was monitored by transrectal palpation and ultrasonography every 3 days during diestrus and daily during estrus. On the first cycle, mares were administered 2500 IU hCG intravenously once a follicle 35 mm was detected. Mares were subsequently inseminated with pooled fresh semen from 2 stallions (1 billion motile sperm) daily until ovulation was detected. An embryo collection procedure was performed 7 days after ovulation. Mares were subsequently administered cloprostenol, and eFSH treatment was initiated. Mares received 12 mg eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time hCG was administered. Mares were inseminated and embryo collection attempts were performed as described previously.

Statistical analysis

In experiment 1, 1-way analysis of variance with F protected LSD was used to analyze quantitative data. Pregnancies per ovulation were analyzed by x² analysis. In experiment 2, number of large follicles, ovulation rate, and embryo recovery rate were compared by Student,'s t-test. Data are presented as the mean S.E.M. Differences were considered to be statistically significant at p < .05, unless otherwise indicated.

Results

In experiment 1, mares treated with 25 mg eFSH twice daily developed a greater number of follicles 35 mm in diameter (p = .001) and ovulated a greater number of follicles (p = .003) than control mares (Table 1). However, the number of pregnancies obtained per mare was not significantly different between the control group and the group receiving 25 mg eFSH (p = .9518). Mares treated with 12 mg eFSH and administered either hCG or deslorelin to induce ovulation also developed more follicles 35 mm (p = .0016 and .0003, respectively) than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles (p = .003) than control mares, whereas the number of ovulations for mares receiving eFSH followed by deslorelin was similar to that of control mares (p = .3463). Pregnancy rate for mares induced to ovulate with hCG was higher (p = .0119) than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of controls (p = .692). Pregnancy rate per ovulation was not significantly different between control mares (54.5%) and mares treated with eFSH followed by hCG (52.9%). The lowest pregnancy rate per ovulation was for mares stimulated with 25 mg eFSH and induced to ovulate with deslorelin. The mean number of days mares were treated with 25 mg or 12 mg of eFSH was 7.8 ± 0.4 and 7.5 ± 0.5 days, respectively. Overall, 80.0% of mares administered eFSH had multiple ovulations compared with 10.3% of control mares.