Persistent breeding-induced endometritis after hysteroscopic insemination in the mare

Low-dose insemination has been proposed to reduce persistent breeding-induced endometritis (PBIE) in mares with delayed uterine clearance (DUC). Others proposed that hysteroscopic insemination induces an exaggerated inflammatory response and should be avoided in DUC mares. The objectives here were to evaluate presence and severity of PBIE in normal and DUC mares after hysteroscopic insemination with fresh semen, and to determine if hysteroscopy could be used in DUC mares without inducing excessive inflammation. Reproductively normal (n = 4) and DUC (n = 5) mares received four treatments in random order: uterine body insemination (UB, 1 × 10(9) spermatozoa, 20 ml), hysteroscopic insemination (HYST, 5 × 10(6) spermatozoa, 0.5 ml), sham hysteroscopic insemination (SHAM, semen extender, 0.5 ml) and hysteroscopic infusion of seminal plasma (SP, 0.5 ml). Significantly more DUC (50%) mares than normal (14%) mares accumulated intrauterine fluid 24 h post-treatment. The difference in fluid accumulation between DUC (40%) mares and normal (7%) mares was also significant 48 h post-treatment. Fluid scores were not significantly different between treatments in normal mares. However, treatments HYST and SHAM resulted in significantly higher fluid scores 24 h but not 48 h post-treatment in DUC mares. There was no effect of treatment or mare group on the percentage and total number of neutrophils in uterine fluid 48 h post-treatment. Percentage of neutrophils was correlated with duration of hysteroscopy in normal mares, with procedures lasting ≥ 9 min associated with PBIE. There was no effect of mare group, treatment or duration of hysteroscopy on pregnancy rate. Hysteroscopy induces a transient inflammation that is not more severe than that after conventional artificial insemination, suggesting no contraindication to its use in DUC mares.     

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).     

Infertility in the brood mare

This communication describes the methods used and results obtained with a 21-year-old Standardbred sire bred, using artificial insemination techniques, to a book of 108 mares during the period 11th September to 16th March, 1979-80. A positive pregnancy test rate of 93.5% was achieved. Those mares becoming pregnant averaged 4.6 inseminations per pregnancy (range 1-13). Barren mares, maiden mares and wet mares, irrespective of age, all achieved highly satisfactory pregnancy rates and it was noted that breeding through the January/February period improved the ultimate success rate markedly. When the official returns for the breeding season under review were returned the sire had a live foal success rate of 84%.     

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.     

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.     

Retained fetal membranes in the mare: A retrospective study

A retrospective study of 3456 deliveries was conducted from the records of four Standardbred broodmare farms where mares were bred by artificial insemination and maintained under close veterinary supervision. Retained fetal membranes (RFM) were observed in 10.6% of the deliveries. Retained fetal membranes occurred more frequently (p < 0.05) after dystocia and in mares which had RFM the previous year. Retained fetal membranes after normal foaling had no significant effect on the reproductive performance (pregnancy rate, pregnancy loss rate, or foaling rate), nor on the general health of the mares, regardless of the duration of RFM (3 to 144 hours). Postfoaling laminitis was not observed. Oxytocin therapy of mares with RFM starting at two hours postpartum significantly reduced the incidence of RFM ≥ 8 hours. Mares with RFM which had received intrauterine antimicrobials between foaling and first breeding had a foaling rate similar to mares with RFM which had not received intrauterine therapy.     

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.     

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.     

Effect of seminal plasma on uterine inflammation, contractility and pregnancy rates in mares

REASONS FOR PERFORMING STUDY: There is conflicting evidence over the role seminal plasma plays in sperm transport and inflammation within the uterus of mares. In in vitro studies, seminal plasma has been shown to reduce polymorphonuclear neutrophil (PMN) function, but the opposite effect on uterine inflammation has been reported in vivo. OBJECTIVES: To study the effect of seminal plasma on uterine contractility, inflammation and pregnancy rates by inseminating mares with low doses of sperm free from seminal plasma (Group 1) and containing seminal plasma (Group 2). METHODS: Synchronised mares were inseminated with 50 x 10(6) sperm in either skim milk extender or seminal plasma. Uterine lavage was performed 6 h after insemination to assess the inflammatory response. The contraction frequency of the uterus was measured over a 4 min period 10 mins and 6 h after insemination, using B-mode ultrasonography. Pregnancy rates were assessed 16 days after insemination. RESULTS: Uterine contractions were less frequent in Group 1 mares inseminated with seminal plasma and significantly more PMNs were found in the lavage fluid of those mares. Pregnancy rates were identical in both groups (62%). CONCLUSIONS: This study provides evidence that seminal plasma decreases uterine contractility and increases the inflammatory response of the uterus to semen. No effect of seminal plasma on pregnancy rates was demonstrated. POTENTIAL RELEVANCE: Mares that develop persistent mating-induced endometritis may have inherently poor uterine contractility and impaired uterine clearance. The presence of seminal plasma during breeding may not be desirable in these mares. The role of seminal plasma in problem mares warrants additional study.     

Effects of time of insemination relative to ovulation on pregnancy rate and embryonic-loss rate in mares

The effects of pre-ovulatory and post ovulatory insemination on pregnancy rate and embryonic-loss rate were studied in 268 mares in two experiments. Within each experiment mares were randomised within replicates as follows: to be inseminated on the day the pre-ovulatory follicle reached 35 mm (pre-ovulatory group), to be inseminated on the day of ovulation (Day 0 group), and to be inseminated on the day after ovulation (Day 1 group). Ultrasonic pregnancy diagnoses were performed on Days 11, 12, 13 and 14 (Experiment 1) and Days 11, 12, 13, 14, 15, 20 and 40 (Experiment 2). Combined for the two experiments, pregnancy rates were different (P less than 0.01) among the three groups. For Experiment 2, pregnancy rate within the pre-ovulatory group was lower (P less than 0.05) for insemination 4 days or more before ovulation than for up to 3 days before ovulation. Pregnancy rate was lower (P less than 0.05) for the Day 0 group than for the pre-ovulatory group inseminated up to 3 days before ovulation. In Experiment 2, ovulation was detected by examinations every 6 h; pregnancy rate was greater (P less than 0.05) for mares inseminated 0 to 6 h after ovulation than for mares inseminated at 18 to 24 h. No pregnancies occurred when mares were inseminated 30 h or more after ovulation. The mean day of first detection of the embryonic vesicle was different (P less than 0.0001) among the three groups. Diameter of embryonic vesicle averaged over Days 11 to 15 also differed (P less than 0.0001) among groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Conception in mares following intrauterine therapy with amikacin

The semisynthetic aminoglycoside amikacin is a potentially valuable antibiotic in broodmare practice due to its efficiency against Gram negative bacteria which have developed resistance to gentamicin. Each of 4 mares conceived when amikacin at 200 ug/ml in buffered extender was used in artificial insemination or as a prebreeding infusion. After intrauterine infusion of 2 g amikacin in 200 ml saline, 4 mares conceived to natural service or insemination with untreated semen after seven hours. In unbuffered saline solutions, amikacin (1 g/100 ml) and gentamicin (1 mg/ml) were spermicidal in vitro.     

Embryo Transfer Efficiency of Quarter Horse Athletic Mares

This study was designed to compare embryo recovery rates and pregnancy rates of athletic and breeding Quarter Horse mares in a tropical warm climate. Thirty-nine barrel racing mares in training and 135 breeding mares as control donors were included. During the training period, the ambient temperature ranged from 31°C to 36°C and the average humidity from 70% to 90%. After the detection of a 35-mm follicle by ultrasound, ovulation was induced with 1 mg of deslorelin acetate (i.m), and insemination was performed 24 hours later with cooled and fresh semen from different fertile stallions. Embryos were collected on day 8 postovulation. The body temperature (rectal) was evaluated from eight athletic donor mares randomly selected from the same studied group. A total of 138 and 657 embryo collections were carried out on training and breeding mares, respectively, with a total of 105 (76%) and 466 (71%) embryos collected (P > .05). Similarly, no differences (P > .05) were observed for the pregnancy rates on day 15 (82/105, 78% vs. 370/466, 79%), and day 40 (73/105, 69% vs. 328/466, 70%) between the training and breeding donor mares. Just after training, the body temperature increased to an average of 39.4°C and the respiratory rate from 14.5 to 35.3 breaths per minute. The results of the present study showed that embryo production from appropriately trained donor mares in good condition were similar to non-athletic broodmares.     

Successful use in horses of deep-frozen semen specimens stored for 18 years

In 1970 semen from a Haflinger-stallion was frozen by the pellet method. 18 years later semen samples were used to inseminate 4 mares. Inseminations were performed shortly after ovulation with a total number of motile spermatozoa between 150 and 636 x 10(6), the percentage of motile spermatozoa being 20% to 40%. Three mares conceived after a single insemination, one mare got pregnant after 4 inseminations during 3 oestrous periods. Meanwhile, 3 foals were born and one of the mares is still pregnant. The results demonstrate that long-term storage of frozen semen in liquid nitrogen does not impair its fertilizing capacity.     

Follicular dynamics after treatment with hCG for ovulation induction in mares

In this study the use of hCG for induction of ovulation is described. Factors such as follicle diameter at the time of administration of hCG (3000 IE hCG i.v.), follicular growth after hCG and the rate of double ovulations were evaluated. A total of 168 mares presented for artificial insemination were used. In 249 estrous periods hCG was given to mares exhibiting standing estrous when a minimum follicle diameter of 30 mm and a well developed edema of the endometrium could be detected by ultrasonography. In nine estrous periods ovulation occurred within 24 hours after hCG. The majority of mares (216; 86.7%) ovulated 24 to 48 hours after hCG and in 24 cases ovulation was delayed beyond 48 hours. Follicle size at the time of hCG administration (30-34 mm, 35-39 mm, > or = 40 mm) had no influence on the percentage of mares ovulating 24 to 48 hours after hCG (89.2%, 87.9%, and 83.7%, respectively). Double ovulations could be observed in 17.7% of estrous periods. The one cycle pregnancy rate was not influenced by follicle size (small 45.9%; medium 41.6%; large 47.5%). Repeated treatments with hCG during successive estrous cycles within one year did not influence the rate of responding to hCG. Mares in standing estrous respond well to hCG if a minimum follicle size of 30 mm and a well developed endometrial folding is present.     

Hysteroscopic insemination of mares with low numbers of nonsorted or flow sorted spermatozoa

The objectives of this study were 1) to compare pregnancy rates resulting from 2 methods of insemination using low sperm numbers and 2) to compare pregnancy rates resulting from hysteroscopic insemination of 5 x 106 nonsorted and 5 x 106 spermatozoa sorted for X- and Y-chromosome-bearing populations (flow sorted). Semen was collected with an artificial vagina from 2 stallions of known acceptable fertility. Oestrus was synchronised (June to July) in 40 mares, age 3-10 years, by administering 10 ml altrenogest orally for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. All mares were given 3000 iu hCG i.v. at the time of insemination to induce ovulation. Mares were assigned randomly to 1 of 3 treatment groups: mares in Treatment 1 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited deep into the uterine horn with the aid of ultrasonography. Mares in Treatment 2 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited onto the uterotubal junction papilla via hysteroscopic insemination. Mares in Treatment 3 (n = 20) were inseminated using the hysteroscopic technique with 5 x 10(6) flow sorted spermatozoa. Spermatozoa were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Pregnancy was determined ultrasonographically at 16 days postovulation. Hysteroscopic insemination resulted in more pregnancies (5/10 = 50%) than did the ultrasound-guided technique (0/10 = 0%; P<0.05) when nonsorted sperm were inseminated. Pregnancy rates were not significantly lower (P>0.05) when hysteroscopic insemination was used for sorted (5/20 = 25%) and nonsorted spermatozoa (5/10 = 50%). Therefore, hysteroscopic insemination of low numbers of flow sorted stallion spermatozoa resulted in reasonable pregnancy rates.     

A field study on reproductive efficiency of mares maintained predominately on native pasture

A field study utilizing 145 mares of various age and breeding status was conducted to determine reproductive performance under native range conditions with only limited supplemental feeding. All mares had an average initial body condition score of 4.5 and a final score of 5.1 by the time breeding was completed and mares returned to pasture. Average foaling rate was 80%, and mares that had not conceived during the previous breeding season had a foaling rate of 94%, which was higher (P<.05) than 74% for lactating mares. Mares 16 years and older had a significantly lower foaling rate (P<.05) than younger mares. Old mares that were lactating at time of breeding had only a 37% foaling rate, which was less (P<.05) than for young lactating mares. The 94 mares bred by natural cover or artificial insemination that actually foaled required 1.43 cycles per conception. Lactating mares in the oldest age group required more cycles per conception (P<.05) than open mares, and these older, lactating mares also required more cycles per conception than younger mares with foals at side. Those mares diagnosed as pregnant or open at 45 days post breeding had a pregnancy rate of 97%. Average pregnancy loss for all mares was 7.7%. These data indicate that lactating mares in moderate body condition tended to skip a breeding season and that a body condition score of 5 was only marginally acceptable, especially in the case of lactating mares. Authors' address: Equine Science Program, Department of Animal Science, Texas Agricultural Extension Service, Texas A&M University, College Station, TX 77843. Technical Article Number 30023, Texas Agr. Exp. Sta.     

Comparison of endometrium before and after repair of third-degree rectovestibular lacerations in mares.

The endometrial response of mares to repair of third-degree rectovestibular lacerations was evaluated. Endometrial biopsy specimens from 8 mares with third-degree rectovestibular laceration were obtained immediately before surgery and from 9 to 15 days after repair. Presurgical endometrial biopsy specimens were classified as category I for 2 mares; category II, attributable to slight endometritis, for 5 mares; and category III, attributable to moderate-to-severe endometritis, for 1 mare. Within 15 days after rectovestibular repair, all endometrial biopsy specimens were classified as category I. Results indicated that mares with third-degree rectovestibular injuries are candidates for breeding by artificial insemination by 2 weeks after repair of the injury.     

Safety of altrenogest in pregnant mares and on health and development of offspring

Fifty-one light-horse mares were utilized to evaluate the safety of an oral progestin, altrenogest, administered throughout gestation on: gestation length, embryonic and fetal loss, periparturient events, health and development of offspring, and future reproductive capabilities of the mares. Pregnancies were established by inseminating mares with 250 × 10⁶ progressively motile spermatozoa from the same stallion every other day throughout estrus or by non-surgical transfer of embryos. Mares were randomly assigned to 1 of 2 treatments upon confirmation of pregnancy on day 20: 1) controls, 2 ml of neobee oil orally per 44.5 kg of body weight; and 2) treated, 2 ml of altrenogest dissolved in neobee oil at a concentration of 2.2 mg/ml orally per 44.5 kg of body weight. Treatments were administered daily from day 20 to 320 of gestation.There were no significant differences between treatment groups for duration of gestation, placental weight, time to placental expulsion and incidence of retained placental membranes. Number of female foals born from altrenogest treated mares (14 of 23) was greater (P<.05) than the number from untreated control mares (4 of 16). Female foals born from altrenogest treated mares had larger clitori (P<.05) than those from control mares. Times to sternal recumbency, standing and nursing were similar for the 2 groups (P>.05). Body weight and height at withers, heart girth circumference and length and width of cannon were measured at time of birth and at 2, 4, 6, 8, 12 and 16 weeks of age. Measurements did not differ (P>05) between treated and control foals for any development parameters.Beginning on day 20 postpartum, mares were teased daily. During estrus, mares were inseminated every other day with 250 × 10⁶ motile spermatozoa. Teasing and/or insemination was continued for 2 cycles or until mares were 35 days pregnant. The number of mares pregnant after 1 cycle and after 2 cycles of insemination was similar (P>.05) for treated and control mares. Nineteen of 21 treated mares and 15 of 16 control mares were pregnant after 2 cycles of insemination. Number of cycles per pregnancy was similar (P>.05) for treated and control mares (1.37 vs 1.13) as was number of days mares exhibited estrus (6.30 vs 6.13). Number of inseminations per cycle did not differ (P>.05) between treated and control mares (2.92 vs 3.00). In summary, there was no effect of treatment with altrenogest from day 20 to 320 of gestation on periparturient events, viability and growth of offspring and subsequent reproductive performance of mares.     

The Effects of Treatment with N-Acetyl Cysteine on Clinical Signs in Persistent Breeding-Induced Endometritis Susceptible Mares

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Endometrial inflammation that persists until embryonic descent ultimately results in early embryonic death. A poor endometrial biopsy grade (IIb or III) has been identified as a risk factor for PBIE. Intrauterine fluid accumulation (>2 cm in depth), pathologic endometrial edema, and elevated intrauterine neutrophil levels are all clinical features of PBIE. Commonly applied treatment options include uterine lavage and oxytocin therapy. N-acetyl cysteine (NAC), a mucolytic used to treat bacterial endometritis in mares, has anti-inflammatory properties and was investigated as a potential treatment for PBIE. A randomized, blinded, cross-over design clinical trial used NAC before breeding in PBIE-susceptible mares (n = 9). Intrauterine infusion of 3.3% NAC was performed 12 hours before insemination, and endometrial cytology and endometrial biopsy samples were obtained at 12 and 60 hours after insemination. Endometrial biopsies were evaluated for the degree of inflammation present. Clinical signs of endometrial edema and intrauterine fluid volumes were assessed by transrectal ultrasound at 12 and then every 24 hours after breeding. Data were analyzed using repeated measures analysis of variance and a Mann Whitney Wilcoxon Test. Treatment with NAC did not improve clinical signs in PBIE-affected mares. However, endometrial biopsies from mares treated with NAC displayed more diffuse and severe neutrophil infiltration than control cycles. Further research using a larger population of mares is required to evaluate the effects of NAC treatment on the endometrium of PBIE-susceptible mares.