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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

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. Procedure—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 ≥ 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%). Conclusion 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. (J Am Vet Med Assoc 2000;217:58–63)

Summary The physiological effect on the equine respiratory tract of recycled phone book paper vs. sawdust bedding was investigated in 12 mares with no clinical signs of respiratory disease. Mares were housed in a 20-stall barn and randomly assigned to one of two treatment groups (Group 1=sawdust; Group 2=phone book paper) for 14 days. After a 14-day turnout, Group 1 was assigned to phone book paper and Group 2 was assigned to sawdust for 14 days using a crossover experimental design. Endoscopic examinations of the upper respiratory tract and trachea along with tracheal washes (TW) were performed on days 0 and 14 of each trial. Cytologic examinations were performed on TW samples. Cell type and laboratory interpretation of TW samples were noted. There was no difference (P>0.05) in cell type between treatment groups within or across trials. Laboratory interpretation indicated no abnormalities in 30 of 48 TW samples. Stall conditions were visually rated and graded daily for drinking water cleanliness, depth of bedding and absorbency, overall cleanliness, ammonia odor, and aerial ammonia levels. The phone book paper bedding was more absorbent (P

Tremendous progress has been made in the development of assisted reproductive techniques that may enhance the reproductive efficiency of the horse. However, techniques that involve the manipulation of oocytes and/or embryos may themselves be detrimental to embryo viability and subsequent development. Therefore, an objective method of assessing viability of embryos before and/or after oocyte/embryo manipulation is desirable. At this time, morphologic evaluation is the most widely used method of determining the viability of equine embryos. Although morphologic assessment of embryo quality will not always accurately predict the survival of individual embryos, it is very useful for predicting the survival of groups of embryos. Other tests that have been used to evaluate equine embryo viability include (1) development during culture in vitro; (2) quantitating metabolism of the fluorescent substrate fluorescein diacetate; (3) quantitating uptake of the fluorescent stain DAPI; and (4) quantitating embryo metabolism. Although these tests offer potential advantages over morphologic assessment alone, their current limitations have prevented their routine use for embryo evaluation. It is likely that as improvements are made in these evaluation methods, they may offer advantages for use alone or in combination to more accurately assess the viability of equine embryos.

The objective of this study was to determine if the primary circulating metabolite of PGF2alpha, 13,14-dihydro-15-keto-PGF2alpha (PGFM), is biologically active and would induce luteolysis in nonpregnant mares. On Day 9 after ovulation, mares (n = 7/group) were randomly assigned to receive: 1) saline control, 2) 10 mg PGF2alpha or 3) 10 mg PGFM in 5 mL 0.9% sterile saline i.m. On Days 0 through 16, blood was collected for progesterone analysis. In addition, blood was collected immediately prior to treatment, hourly for 6 h, and then at 12 and 24 h after treatment for progesterone and PGFM analysis; PGFM was measured to verify that equivalent amounts of hormone were administered to PGF2alpha- and PGFM-treated mares. Mares were considered to have undergone luteolysis if progesterone decreased toor = 1.0 ng/mL within 24 h following treatment. Luteolysis was induced in 0/7 control, 7/7 PGF2alpha-treated, and 0/7 PGFM-treated mares. There was no difference (P0.1) in the occurrence of luteolysis in control and PGFM-treated mares. More (P0.001) PGF2alpha-treated mares underwent luteolysis than control or PGFM-treated mares. There was no difference (P0.1) in progesterone concentrations between control and PGFM-treated mares on Days 10 through 16. Progesterone concentrations were lower (P0.01) on Days 10 through 14 in PGF2alpha-treated compared with control and PGFM-treated mares. There was no difference (P0.05) in PGFM concentrations between PGF2alpha- and PGFM-treated mares; PGFM concentrations in both groups were higher (P0.001) than in control mares. These results do not support the hypothesis that PGFM is biologically active in the mare, since there was no difference in corpora luteal function between PGFM-treated and control mares.

Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy rates. Several ejaculates from each of 5 stallions were frozen in a skim milk-egg yolk based freezing medium at 2 spermatozoal concentrations in 0.5-mL polyvinyl-chloride straws. Half of each ejaculate was frozen at 400 x 10(6) cells/mL and half at 1,600 x 10(6) cells/mL. Insemination doses were based on post-thaw spermatozoal motility and contained approximately 320 x 10(6) (320 to 400) motile spermatozoa or approximately 800 x 10(6) (800 to 900) motile spermatozoa. Sixty-three mares were assigned to 1 of 4 spermatozoal treatments (1--low spermatozoal number, low concentration; 2--low spermatozoal number, high concentration; 3--high spermatozoal number, low concentration; 4--high spermatozoal number, high concentration) and were inseminated daily. Post-thaw spermatozoal motility was similar for cells frozen at both spermatozoal concentrations (P0.1). One-cycle pregnancy rates were 15, 40, 28 and 33%, respectively, for Treatments 1, 2, 3 and 4. Packaging spermatozoa at the high concentration tended to increase pregnancy rates vs packaging at the low concentration (37 vs 22%; P = 0.095). Furthermore, when the lower spermatozoal number was used, there tended (P0.1) to be a higher pregnancy rate if spermatozoa were packaged at the higher concentration. There was no increase in pregnancy rates when higher numbers of motile spermatozoa were inseminated (27 vs 31%; P0.1). Based on these results, a single 0.5-mL straw dose containing 800 x 10(6) spermatozoa should be used and each insemination dose should contain approximately 320 x 10(6) motile spermatozoa. Fertility trials utilizing other freezing extenders are necessary before recommending a single 0.5-mL insemination dose for all freezing extenders.

We recently reported the birth of the first clone of an equine species, a mule, which was produced using a fetal fibroblast cell line (Woods GL et al., 2003 Science 301, 1063). Since the birth of the first foal, two more identical cloned mule foals have been born. All three foals were delivered spontaneously without assistance, and have been healthy and vigorous since birth. Even more recently, the birth of a horse foal cloned from an adult fibroblast cell line was reported (Touchette N, 2003 Nature 424, 635). Despite these successes, the efficiency of equine nuclear transfer (NT) continues to be very low. The objective of this study was to use NT to clone adult horses using cumulus cells. Cumulus-oocyte complexes used for NT were obtained using transvaginal ultrasound-guided follicle aspiration (TVA) 24hrs after hCG treatment; oocytes were used as cytoplasts, while cumulus cells (from one of three different mares) were used as donor cells. Cumulus cells were recovered from TVA fluid, washed two times by suspension in PB1 medium (Whittingham DG, 1974 J. Reprod. Fertil. 37, 159–162), followed by centrifugation (200g) and placement in Glasgow MEM BHK-21 containing 10% FBS. Nuclear transfer procedures were performed as described (Woods GL et al., 2003 Science 301, 1063). Immediately following NT and activation procedures, cloned embryos were surgically transferred to the oviduct of recipient mares (n=2 to 5 embryos/recipient) that had ovulated within 24hrs prior to the transfer. An initial pregnancy examination was performed between Days 14 and 16 (Day 0=surgery); subsequent examinations were then performed at approximately weekly intervals. A total of 136 follicles were aspirated in 96 mares, from which 72 oocytes were recovered (53%). Sixty-two cloned embryos were subsequently transferred to recipient mares, which resulted in 7 (11.3%) ultrasonographically-detectable pregnancies. Cumulus cells from Mare 160 tended (P=0.08) to result in more pregnancies than cumulus cells from Mare 221 (4/17 v. 1/25, respectively). All seven cloned pregnancies underwent spontaneous pregnancy loss between Days 16 and 80. An embryo-proper and heartbeat were detected in three conceptuses. Of four conceptuses in which an embryo-proper was not observed, three did not develop past Day 24; therefore, they were lost before the time at which an embryo-proper generally becomes readily apparent. One conceptus developed to Day 28, yet still failed to form an embryo-proper. There were no premonitory signs of impending embryonic loss in the conceptuses that did not develop an embryo-proper; the conceptus was simply not evident at the subsequent examination. Signs of impending embryonic loss were observed in the three conceptuses in which an embryo-proper was observed, and included: (1) loss of embryonic heartbeat, (2) disorganization of the conceptus membranes, and (3) increased echogenicity of conceptus fluids. One or more of these signs were observed in all three conceptuses prior to pregnancy loss. To our knowledge, this is the first report documenting the establishment of cloned horse pregnancies produced using adult cumulus cells.