Identification of heat shock protein 10 within the equine embryo, endometrium, and maternal peripheral blood mononuclear cells.

Early pregnancy factor has been identified as a 10-kDa extracellular homolog of heat shock protein 10 (Hsp10). Hsp10 has been detected during early pregnancy in serum of mice, sheep, pigs, horses, cows, and humans by the rosette inhibition test. Hsp10 has also been associated with several neoplastic and autoimmune diseases. The goal of the present study was to determine if Hsp10 could be detected in the early equine embryo through the use of immunohistochemistry and quantitative real-time PCR. Additionally, analysis of systemically harvested peripheral blood mononuclear cells (PBMCs) from both pregnant and nonpregnant mares was evaluated to determine expression levels of HSP10. Embryos were collected from Quarter Horse mares by uterine lavage at either 8 or 25 days after ovulation. Collection and separation of PBMCs occurred on Day 8 for both pregnant and nonpregnant mares. Immunohistochemistry revealed cytoplasmic localization of HSP10 throughout the single layer of ectodermal cells forming the trophoblast in Day-8 embryos. Day-25 embryos demonstrated intense localization focally along the apical border of ectodermal cells forming the trophoblast layer of the developing chorion. There was no nuclear staining in either embryonic population. Quantitative real-time PCR detected the presence of mRNA for HSP10 in both 8- and 25-day equine embryos. Day-25 embryos exhibited an elevated degree of expression (P = 0.006) compared with the 8-day embryos for HSP10. Endometrial samples did not display any significant difference in degree of expression for HSP10 (P = 0.10). Finally, PBMCs from pregnant mares demonstrated elevated (P = 0.03) expression of HSP10 compared to the nonpregnant mares on Day 8 of the estrous cycle. This study confirmed the presence of HSP10 protein and mRNA expression of HSP10 in equine embryos at two maturation stages. Additionally, the presence of increased gene expression within PBMCs of pregnant mares suggests communication, possibly leading to necessary immunomodulatory effects between the embryo and mare.

Detection of bacteraemia and host response in healthy neonatal foals.

REASONS FOR PERFORMING THE STUDY: Neonatal sepsis is a common problem in foals and is a primary cause of death in the post natal period. Transient bacteraemia and subsequent host responses have not been described in the equine neonate. OBJECTIVES: The primary objective of this study was to determine if transient bacteraemia occurs in foals within the first 72 h of life. Additional objectives included description of bacterial organisms associated with transient bacteraemia and concurrent cytokine gene expression in healthy foals. STUDY DESIGN: Prospective observational study in healthy foals. METHODS: Blood was aseptically collected for bacterial culture from observed spontaneously born foals at birth and 1, 2, 3, 4, 8, 12, 24, 48 and 72 h following birth. Samples taken at birth, 4, 12, 24, 48 and 72 h were analysed for interferon gamma (IFNγ), interleukin (IL)-1, IL-2, IL-6, IL-8, IL-10, IL-18 and monocyte chemotactic protein 1 (MCP1) cytokine gene expression quantified by RT-PCR. RESULTS: Bacteria were cultured from 9 of 70 samples submitted for blood culture. The positive samples were from 4 of the 7 foals, all of which remained healthy throughout and subsequent to the study. All positive blood cultures were from blood samples obtained at 12 h of age or earlier and IL-10 elevation coincided with positive blood cultures in healthy foals. Cytokine gene expression fluctuated with age. CONCLUSIONS: Positive blood cultures suggest transient bacteraemia may occur in healthy foals early in the post natal period. Age corrected normal values may be necessary to interpret cytokine concentration in diseased populations.

Parturition, dystocia and foal survival: a retrospective study of 1047 births.

REASON FOR PERFORMING STUDY: An understanding of the normal events of foaling, causes of dystocia and clinical outcomes is important for equine practitioners. OBJECTIVES: The goals of the present study were to: 1) evaluate factors that influence gestation length; 2) report duration of Stage II labour; 3) determine the frequency of dystocia and premature placental separation; and 4) determine the relationship between problems at foaling and foal survival. MATERIALS AND METHODS: Foaling records of 1047 mare births were evaluated. RESULTS: The average gestation length was 342.7 days [corrected] +/- 0.4 days, with no effect of mare age or breed observed. Mares carrying male fetuses had a longer gestation (P < or = 0.001) than mares carrying female fetuses. A majority (52.8%) of mares foaled at night between 2000 h and 0200 h when the facility was quiet. Dystocia occurred in 10.1% of all births and the incidence rate was higher in Thoroughbred mares than in Quarter Horse mares. The most common cause of dystocia was abnormalities of fetal posture. A delay in foal delivery beyond 40 min of Stage II of labour was associated with a significant increase in foal mortality. In addition, an increase in foal morbidity and mortality was noted when the interval from birth to standing or birth to nursing was prolonged. CONCLUSION: Early detection and rapid appropriate intervention are critical to foal survival in an equine dystocia. POTENTIAL RELEVANCE: Equine veterinarians should counsel horse owners that early recognition of a foaling problem and rapid, appropriate intervention are critical to the survival of a foal.

The effects of dexamethasone and prednisolone on pituitary and ovarian function in the mare.

REASONS FOR PERFORMING STUDY: Persistent mating induced endometritis is among the most common causes of infertility in the mare. Recently, improved pregnancy rates have been reported when corticosteroids were administered to 'problem mares' specifically, to modulate the post mating inflammatory response; however, the effect of treatment on pituitary and ovarian function requires further study. OBJECTIVES: To evaluate the effects of prolonged treatment with glucocorticoids on pituitary and ovarian function. METHODS: Eighteen cycling Quarter Horse mares in early oestrus were assigned randomly to one of 3 treatment groups: dexamethasone 0.05 mg/kg bwt i.v. twice a day, prednisolone 0.5 mg/kg per os twice a day, or placebo for 5 days. Mares were examined by ultrasound daily to evaluate reproductive function. Blood samples were collected daily to measure luteinising hormone (LH), progesterone and cortisol levels. RESULTS: Dexamethasone treatment caused greater (P<0.05) suppression of endogenous cortisol concentration (9.4 +/- 1.1 ng/ml) compared to prednisolone- (41.9 +/- 4.0 ng/ml) or placebo-treated mares (32.4 +/- 3.8 ng/ml). After 24 h, mares treated with dexamethasone exhibited lower uterine oedema scores than prednisolone- or placebo-treated mares. An ovulation rate of 40% was observed in dexamethasone-treated mares (2/5) compared to 83% for prednisolone (5/6) and 100% for placebo-treated (6/6) mares. An absence of a LH surge was noted in 3 of 5 dexamethasone-treated mares and one of 6 prednisolone-treated mares. CONCLUSIONS: Repeated administration of dexamethasone to mares in oestrus is associated with decreased uterine oedema, suppression of LH and a high rate of ovulation failure. It is recommended that dexamethasone treatment is limited to only 1 or 2 days and that a lower dose is considered in the management of persistent mating induced endometritis to avoid potential adverse affects on reproductive function.

Evaluation of a veterinary glucometer for use in horses.

BACKGROUND: Glucose assessment and regulation are important factors in the treatment of hospitalized horses and foals. HYPOTHESIS/OBJECTIVES: The purpose of this study was to compare glucose measurement by a veterinary glucometer, adjusted by code for use in horses and foals, to a reference chemistry analyzer. It was hypothesized that the veterinary glucometer and reference analyzer would yield similar results and that interpretation of glucose values obtained from a veterinary glucometer would result in clinically appropriate decisions. ANIMALS: Fifty blood samples from adult horses and 50 blood samples from neonatal foals admitted to the Colorado State University Veterinary Hospital or Equine Reproduction Laboratory for evaluation. METHODS: Glucose concentrations from fresh whole blood samples were evaluated in duplicate with a veterinary glucometer and these values were compared with those obtained with a reference plasma chemistry analyzer. The accuracy of glucometer measurement was evaluated with a Clarke error grid. RESULTS: The veterinary glucometer accurately measured whole blood glucose concentrations in both horses and foals when compared with a reference plasma chemistry analyzer. Nearly 97% of the glucometer values obtained in this study would have resulted in appropriate clinical decisions based on the Clarke error grid analysis. CONCLUSIONS AND CLINICAL IMPORTANCE: The veterinary glucometer evaluated has potential utility for point-of-care whole blood glucose evaluation in both horses and foals.

Effect of dehydration prior to cryopreservation of large equine embryos.

Cryopreservation of equine embryos>300microm in diameter results in low survival rates using protocols that work well for smaller equine embryos. These experiments tested the potential benefit of incorporating a dehydration step prior to standard cryopreservation procedures. Forty-six, day 7-8, grade 1, equine embryos 300-1350microm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6M galactose, 10min in 1.5M glycerol, slow freeze (n=21); (B) 10min in 1.5M glycerol, slow freeze (n=15); (C) 2min in 0.6M galactose, 10min in 1.5M glycerol, followed by exposure to thaw solutions, then culture medium (n=5); (D) transferred directly to culture medium (n=5). Frozen embryos were thawed and subjected to a three-step cryoprotectant removal. Five embryos from each treatment were evaluated morphologically after 24 and 48h culture (1=excellent, 5=degenerate/dead). All treatments had at least 4/5 embryos with a quality score >or=3 at these time points except treatment B (2/5 at 24h, 1/5 at 48h). Subsequent embryos from treatment A (n=16) or B (n=10) were matched in sets of two for size and treatment, thawed, and immediately transferred in pairs to 13 recipients. Only two recipient mares were pregnant; one received two 400microm embryos from treatment A, and the other one 400 and one 415microm embryo from treatment B. There was no advantage of incorporating a 2min dehydration step into the cryopreservation protocol for large equine embryos.

eFSH in clinical equine practice.

Equine follicle stimulating hormone (eFSH) has been used to induce follicular development in transitional mares and problem acyclic mares, as well as superovulate cycling mares. The most efficacious protocol is to administer 12.5 mg eFSH, intramuscularly, twice daily beginning 5 to 7 days after ovulation when the diameter of the largest follicle is 20 to 25 mm. Prostaglandins are to be administered on the second day of eFSH therapy. Treatment with eFSH is continued for 3 to 5 days until follicle(s) are >or=35 mm in diameter. The mare is subsequently allowed to 'coast' for 36 h, after which human chorionic gonadotropin is administered to induce ovulation.

Evaluation of three equine FSH superovulation protocols in mares.

Superovulation could potentially increase embryo recovery for immediate transfer or cryopreservation. The objectives were to evaluate the effect of pretreatment with progesterone and estradiol (P+E) on follicular response to eFSH and compare doses of eFSH and ovulatory agents on follicular development and ovulation in mares. In Experiment 1, 40 mares were assigned to one of four treatment groups. Group 1 consisted of untreated controls. Group 2 mares were administered eFSH without pretreatment with P+E. Group 3 mares were administered P+E for 10 days starting in mid-diestrus followed by eFSH therapy. Group 4 mares were administered P+E for 10 days followed by eFSH therapy. All treated mares were administered 12.5mg eFSH twice daily and prostaglandins were given on the second day of eFSH therapy. Mares were bred with fresh semen the day of hCG administration and with cooled semen the following day. The numbers of preovulatory follicles and ovulations were lower for mares treated with P+E prior to eFSH treatment. Pretreatment with P+E in estrus also resulted in a lower embryo recovery rate per ovulation compared to the other two eFSH treatment groups. In Experiment 2, two doses of eFSH (12.5 and 6.25mg) and two ovulation-inducing agents (hCG and deslorelin) were evaluated. The number of preovulatory follicles was greater for mares given 12.5mg of eFSH compared to mares given 6.25mg. Number of ovulations was greatest for mares given 12.5mg of eFSH twice daily followed by administration of hCG. Embryo recovery per flush was similar among treatment groups, but the percent of embryos per ovulation was higher for mares given the low dose of eFSH. In summary, there was no advantage to giving P+E prior to eFSH treatment. In addition, even though the lower dose of eFSH resulted in fewer ovulations, embryo recovery per flush and embryo recovery per ovulation were similar or better for those given the lower dose of eFSH.

Superovulation in mares.

Embryo recovery from single ovulating mares is approximately 50 per cent per estrous cycle. Superovulation could be used to increase embryo recovery and provide extra embryos for embryo freezing. This review addresses some historical approaches to superovulation, as well as examines factors that affect the response of mares to equine FSH. eCG, GnRH and inhibin vaccines have been of limited success in stimulating multiple ovulation. Numerous studies have shown that injection of equine pituitary extract (EPE) will result in three to four ovulations per estrous cycle and two embryos. A purified, standardized EPE preparation (eFSH) also results in a similar response to EPE. Factors affecting the response to EPE and eFSH include day of initial treatment, size of largest follicle at initial treatment and frequency of injection. Embryos from single ovulating, untreated mares and eFSH-treated mares provide similar pregnancy rates upon nonsurgical transfer. Five to 7 days of eFSH treatment also has been shown to hasten the first ovulation of the breeding season. Potential problems after eFSH injections include anovulatory or luteinized follicles and overstimulation. Studies are needed to further evaluate the criteria for initiation of treatment and to determine how to increase ovulation rate without decreasing embryo recovery per ovulation.

Methanol as a cryoprotectant for equine embryos.

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.

Embryo technologies in the horse.

Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 degrees C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts <300 microm can be slowly cooled or vitrified with acceptable pregnancy rates after transfer. The majority of equine embryos are collected from single ovulating mares, as there is no commercially available product for superovulation in equine. However, pituitary extract, rich in FSH, can be used to increase embryo recovery three- to four-fold. Similar to human medicine, assisted reproductive techniques have been developed for the older, subfertile mare. Transfer of in vivo-matured oocytes from young, healthy mares into a recipient's oviduct results in a 70-80% pregnancy rate compared with a 30-40% pregnancy rate when the oocytes are from older, subfertile mares. This procedure can also be used to evaluate in vitro maturation systems. In vitro production of embryos is still quite difficult in the horse. However, intracytoplasmic sperm injection (ICSI) has been used to produce several foals. Cleavage rates of 60% and blastocyst rates of 30% have been reported after ICSI of in vitro-matured oocytes. Gamete intrafallopian tube transfer (GIFT) is a possible treatment for subfertile stallions. Transfer of in vivo-matured oocytes with 200,000 sperm into the oviduct of normal mares resulted in a pregnancy rate of 55-82%. Oocyte freezing is a technique that has proven difficult in most species. However, equine oocytes vitrified in a solution of ethylene glycol, DMSO, and Ficoll and loaded onto a cryoloop resulted in three pregnancies of 26 transfers and two live foals produced. Production of a cloned horse appears to be likely, as several cloned pregnancies have recently been produced.

Removal of deslorelin (Ovuplant) implant 48 h after administration results in normal interovulatory intervals in mares.

Deslorelin implants, approved for use in inducing ovulation in mares, have been associated with prolonged interovulatory intervals in some mares. Administration of prostaglandins in the diestrous period, following a deslorelin-induced ovulation, has been reported to increase the incidence of delayed ovulations. The goals of the present study were: (1) to determine the percentage of mares given deslorelin that experience delayed ovulations with or without subsequent prostaglandin treatment, and (2) to determine if removal of the implant 48 h after administration would effect the interval to subsequent ovulation. We considered interovulatory intervals to be prolonged if they were greater than the mean +/- 2 standard deviation (S.D.) of the control group in study 1 and the hCG group in study 2. In study 1, we retrospectively reviewed reproduction records for 278 mares. We either allowed the mare to ovulate spontaneously or induced ovulation using deslorelin acetate implants or hCG. We administered prostaglandin intramuscularly, 5-9 days after ovulation in selected mares in each group. A higher percentage of mares which were induced to ovulate with deslorelin and given prostaglandins had a prolonged interovulatory interval (23.5%; n = 16), as compared to deslorelin-treated mares that did not receive prostaglandins (11.1%; n = 5). In study 2, we induced ovulation in mares with hCG (n = 47), a subcutaneous deslorelin implant via an implanting device provided by the manufacturer (n = 28), or a deslorelin implant via an incision in the neck (n = 43) and we removed the implant 48 h after administration. We administered prostaglandin to all mares 5-9 days after ovulation. In study 2, mares from which the implant was removed had a normal ovulation rate and none had a prolonged interval to ovulation. Administration of prostaglandin after deslorelin treatment was associated with a longer interval from luteolysis to ovulation than that found in mares not treated with deslorelin. Prostaglandin administration during diestrus may have exacerbated the increased interval to ovulation in deslorelin-treated mares. We hypothesize that prolonged secretion of deslorelin from the implant was responsible for the extended interovulatory intervals.

Strategies to improve the ovarian response to equine pituitary extract in cyclic mares.

Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation; (3) administration of EPE twice daily in successively decreasing doses. In the first experiment, 50 mares were randomly assigned to one of four treatment groups. Mares received (1) 25 mg EPE once daily; (2) 50 mg EPE once daily; (3) 12.5 mg EPE twice daily; or (4) 25 mg EPE twice daily. All mares began EPE treatment 5 days after detection of ovulation and received a single dose of cloprostenol sodium 7 days postovulation. EPE was discontinued once half of a cohort of follicles reached a diameter of >35 mm and hCG was administered. Mares receiving 50 mg of EPE once daily developed a greater number (P = 0.008) of preovulatory follicles than the remaining groups of EPE-treated mares, and more (P = 0.06) ovulations were detected for mares receiving 25 mg EPE twice daily compared to those receiving either 25 mg EPE once daily and 12.5 mg EPE twice daily. Embryo recovery per mare was greater (P = 0.05) in the mares that received 12.5 mg EPE twice daily than those that received 25 mg EPE once daily. In Experiment 2, 20 randomly selected mares received either 25 mg EPE twice daily beginning 5 days after a spontaneous ovulation, or two doses of a GnRH-a agonist upon detection of a follicle >35 mm and 25 mg EPE twice daily beginning 5 days after ovulation. Twenty-four hours after administration of hCG, oocytes were recovered by transvaginal aspiration from all follicles >35 mm. No differences were observed between groups in the numbers of preovulatory follicles generated (P = 0.54) and oocytes recovered (P = 0.40) per mare. In Experiment 3, 18 mares were randomly assigned to one of two treatment groups. Then, 6-11 days after ovulation, mares were administered a dose of PGF2, and concomitantly began twice-daily treatments with EPE given in successively declining doses, or a dose of PGF2alpha, but no EPE treatment. Mares administered EPE developed a higher (P = 0.0004) number of follicles > or = 35 mm, experienced more (P = 0.02) ovulations, and yielded a greater (P = 0.0006) number of embryos than untreated mares. In summary, doubling the dose of EPE generated a greater ovarian response, while increasing the frequency of treatment, but not necessarily the dose, improved embryo collection. Additionally, pretreatment with a GnRH-a prior to ovarian stimulation did not enhance the response to EPE or oocyte recovery rates.

Deslorelin acetate (Ovuplant) therapy in cycling mares: effect of implant removal on FSH secretion and ovarian function.

Following induction of ovulation with deslorelin acetate (Ovuplant), gonadotrophin concentrations are reduced in the subsequent cycle, leading to increased interovulatory intervals in some mares. This study determined whether implant removal after 2 days prevented the decrease in gonadotrophin concentrations and follicular growth during the ensuing cycle. Twenty-four mares were randomised equally into 3 groups. Group 1 ovulated spontaneously, Groups 2 and 3 received the deslorelin implant to induce ovulation. Two days after treatment, the implant was removed from Group 3. On Day 10 postovulation, FSH was lower (P = 0.009) in Group 2, but not different between Groups 1 and 3. Follicular diameter on Day 14 was less (P<0.05) in Group 2 (19.0 +/- 2.1 mm) than in Groups 1 and 3 (36.6 +/- 2.5 and 30.5 +/- 2.0 mm, respectively). Interovulatory interval was longer (P<0.05) for Group 2 (25.8 +/- 2.9 days) compared to Groups 1 and 3 (18.5 +/- 0.7 and 19.4 +/- 0.3 days, respectively). Removal of the deslorelin implant eliminated the decreased FSH secretion and the increased interovulatory interval associated with implant administration. Therefore, it is recommended that the implant be removed after ovulation is detected to prevent the occurrence of a prolonged interovulatory interval.

Ovarian superstimulatory response and embryo production in mares treated with equine pituitary extract twice daily.

Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Group 2). In Experiment 1, more (P < 0.05) follicles > or = 35 mm were detected in mares treated with EPE twice daily (6.1 +/- 3.1) than in mares treated once a daily (2.0 +/- 0.6). In a second experiment, the embryo recovery rates of mares given the two EPE protocols used in Experiment 1 were compared. The number of ovulations per mare was higher (P < 0.05) for mares treated twice-daily (7.1 +/- 5.1, range 3 to 18) than for mares treated once daily (2.4 +/- 1.8, range 1 to 6). The number of embryos produced per mare was higher (P < 0.05) in mares in Group 2 (3.5) than in Group 1 (1.6). Although it is not clear whether the increased ovulation rate is due specifically to dose or frequency, twice-daily administration of a high dose of EPE significantly improved follicular development, ovulation and embryo recovery over the standard treatment of once-daily injection.

Effect of deslorelin acetate on gonadotropin secretion and ovarian follicle development in cycling mares.

OBJECTIVE: To evaluate gonadotropin secretion and ovarian function after administration of deslorelin acetate to induce ovulation in mares. DESIGN: Randomized controlled trial. ANIMALS: 16 healthy mares with normal estrous cycles. PROCEDURE: 8 control mares were allowed to ovulate spontaneously, whereas 8 study mares received deslorelin to induce ovulation when an ovarian follicle > 35 mm in diameter was detected. Follicle development and serum concentrations of gonadotropins were monitored daily during 1 estrous cycle. Pituitary responsiveness to administration of gonadotropin-releasing hormone (GnRH) was evaluated 10 days after initial ovulation. RESULTS: Interovulatory intervals of mares treated with deslorelin (mean +/- SD, 25.6 +/- 2.6 days) were longer than those of control mares (22.9 +/- 1.8 days). Diameter of the largest follicle was significantly smaller during 2 days of the diestrous period after ovulation in deslorelin-treated mares than in control mares. Concentrations of follicle-stimulating hormone (FSH) were lower in deslorelin-treated mares on days 5 through 14 than in control mares. Concentrations of luteinizing hormone were not different between groups during most of the cycle. Gonadotropin release in response to administration of GnRH was lower in mares treated with deslorelin acetate than in control mares. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of deslorelin was associated with reduction in circulating concentrations of FSH and gonadotropin response to administration of GnRH during the estrous cycle. Low concentration of FSH in treated mares may lead to delayed follicular development and an increased interovulatory interval.

Fertility of mares after unilateral laparoscopic tubal ligation.

OBJECTIVE: To develop a technique for laparoscopic tubal (oviductal) ligation and to evaluate pregnancy rates for mares that ovulated ipsilateral or contralateral to the ligated oviduct. STUDY DESIGN: Randomized prospective clinical trial comparing pregnancy rates after unilateral laparoscopic tubal ligation. ANIMALS: Twelve mares of light horse breeds. METHODS: One oviduct in each of 6 mares was surgically ligated with a laparoscopic technique; 6 other mares served as nonligated controls. Mares with unilateral tubal ligations (UTL) were inseminated with 500 million progressively motile sperm during 1 cycle when the dominant follicle was ipsilateral to the ligation site and 1 cycle when the dominant follicle was contralateral to the ligation site. Control mares were bred during 2 cycles regardless of the side of the dominant follicle. Pregnancy examinations were performed on days 12, 14, and 16 after ovulation by transrectal ultrasonography. RESULTS: None of the mares became pregnant when ovulations occurred from the ovary adjacent to the ligated oviduct. All 6 mares became pregnant on the first cycle when an ovulation occurred from the opposite ovary. Control mares became pregnant on 10 of 12 cycles (83.3 %). CONCLUSIONS: UTL was completely effective in preventing pregnancy when ovulation occurred ipsilateral to the ligation site. The surgical procedure did not interfere with the establishment of pregnancy when ovulation occurred from the contralateral ovary. CLINICAL RELEVANCE: UTL may be a clinically useful procedure for preparing a recipient mare for gamete intrafallopian transfer. The recipient mare could be allowed to ovulate and UTL would prevent fertilization of her oocyte but would not interfere with normal corpus luteum formation. The donor oocyte could be placed into the oviduct contralateral to the UTL site.

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer.

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.

Diagnosis and management of abnormal embryonic development characterized by formation of an embryonic vesicle without an embryo in mares.

OBJECTIVE: To determine the incidence, ultrasonographic characteristics, and risk factors associated with embryonic development characterized by formation of an embryonic vesicle without an embryo in mares. DESIGN: Prevalence survey. ANIMALS: 159 pregnant mares. PROCEDURES: From 1994 to 1998, mares between 11 and 40 days after ovulation with normal and abnormal embryonic development were examined ultrasonographically, and characteristics of each conceptus were recorded. RESULTS: The incidence of abnormal embryonic development in mares characterized by formation of an embryonic vesicle without an embryo was 7/159 (4.4%) during the 5 breeding seasons. Age and breed of mare or type of semen used did not differ for mares with normal and abnormal embryonic development. The percentage of mares in which the conceptus was undersized during > or = 1 examination was significantly higher for mares with abnormal conceptuses (5/7), compared with mares with normal conceptuses (2/147; 1.4%). The percentage of examinations during which the conceptus was undersized was significantly higher for abnormal conceptuses (12/27; 44.4%), compared with normal conceptuses (4/448; 0.9%). CONCLUSIONS AND CLINICAL RELEVANCE: To diagnose an embryonic vesicle without an embryo, mares should be examined by use of transrectal ultrasonography on day 25 after ovulation. When an embryo cannot be identified at that time, mares should be reexamined at intervals of 1 to 3 days until day 30. Because undersized conceptuses are more likely to be abnormal, development of undersized conceptuses should be monitored closely.

Insemination of mares with low numbers of either unsexed or sexed spermatozoa.

Two experiments were conducted to determine pregnancy rates in mares inseminated 1) with 5, 25 and 500 x 10(6) progressively motile spermatozoa (pms), or 2) with 25 x 10(6) sex-sorted cells. In Experiment 1, mares were assigned to 1 of 3 treatments: Group 1 (n=20) was inseminated into the uterine body with 500 x 10(6) pms. Group 2 (n=21) and Group 3 (n=20) were inseminated into the tip of the uterine horn ipsilateral to the preovulatory follicle with 25 and 5 x 10(6) pms, respectively. Mares in all 3 groups were inseminated either 40 (n=32) or 34 h (n=29) after GnRH administration. More mares became pregnant when inseminated with 500 x 10(6) (18/20 = 90%) than with 25 x 10(6) pms (12/21 = 57%; P<0.05), but pregnancy rates were similar for mares inseminated with 25 x 10(6) vs 5 x 10(6) pms (7/20 = 35%) (P>0.1). In Experiment 2, mares were assigned to 1 of 2 treatments: Group A (n=11) was inseminated with 25 x 10(6) spermatozoa sorted into X and Y chromosome-bearing populations in a skimmilk extender. Group B (n=10) mares were inseminated similarly except that spermatozoa were sorted into the skimmilk extender + 4% egg yolk. Inseminations were performed 34 h after GnRH administration. Freshly collected semen was incubated in 224 microM Hoechst 33342 at 400 x 10(6) sperm/mL in HBGM-3 for 1 hr at 35 degrees C and then diluted to 100 x 10(6) sperm/mL for sorting. Sperm were sorted by sex using flow cytometer/cell sorters. Spermatozoa were collected at approximately 900 cells/sec into either the extender alone (Group A) or extender + 4% egg yolk (Group B), centrifuged and suspended to 25 x 10 sperm/mL and immediately inseminated. Pregnancy rates were similar (P>0.1) between the sperm treatments (extender alone = 13/10, 30% vs 4% EY + extender = 5/10, 50%). Based on ultrasonography, fetal sex at 60 to 70 d correlated perfectly with the sex of the sperm inseminated, demonstrating that foals of predetermined sex can be obtained following nonsurgical insemination with sexed spermatozoa.