Autogenous transfer of intracytoplasmic sperm injection-produced equine embryos into the uterus of the oocyte donor during the same oestrous cycle.

The clinical use of intracytoplasmic sperm injection (ICSI) in horses usually involves the transfer of embryos into recipient mares, resulting in substantial cost increases. This is essential when subfertile mares are oocyte donors; but some donors are fertile, with ICSI compensating for limited or poor-quality spermatozoa. Fertile oocyte donors could carry pregnancies, eliminating the need for a recipient. We assessed the potential of using oocyte donors as recipients for their own ICSI-produced embryos during the same cycle. Donors in oestrus and with large dominant follicles were administered ovulation-inducing compounds to cause follicle and oocyte maturation. Maturing oocytes were collected, cultured and fertilised using ICSI. At 6 or 7 days after ICSI, developing blastocysts were transferred into respective donors' uteri, and pregnancy rates were determined. Twenty follicles were aspirated from nine mares and 12 oocytes were collected. After ICSI, 10 of the 12 oocytes (83%) cleaved, and eight (67% of injected oocytes) developed into blastocysts for transfer. Five pregnancies resulted from the eight transferred embryos (pregnancy rate 62% per embryo and 42% per sperm-injected oocyte). Following this synchronisation regime, ICSI-produced embryos can be transferred into oocyte donors' uteri during the same cycle, allowing donors to carry pregnancies after assisted fertilisation.

Effects of age and equine follicle-stimulating hormone (eFSH) on collection and viability of equine oocytes assessed by morphology and developmental competency after intracytoplasmic sperm injection (ICSI).

Young (4 to 9 yr) and old (>or=20 yr) mares were treated with equine follicle-stimulating hormone (eFSH), and oocytes were collected for intracytoplasmic sperm injections (ICSI). Objectives were to compare: (1) number, morphology and developmental potential of oocytes collected from young v. old mares from cycles with or without exogenous eFSH and (2) oocyte morphology parameters with developmental competence. Oocytes were collected from preovulatory follicles 20 to 24 h after administration of recombinant equine LH and imaged before ICSI for morphological measurements. After ICSI, embryo development was assessed, and late morulae or blastocysts were transferred into recipients' uteri. Cycles with eFSH treatment resulted in more follicles (1.8 v. 1.2) and more recovered oocytes (1.1 v. 0.8) than those without eFSH. Age and eFSH treatment did not effect cleavage, blastocyst and pregnancy rates. Treatment with eFSH had no effect on oocyte morphology, but age-associated changes were observed. In old mares, zona pellucidae (ZP) were thinner than in young mares, and perivitelline space and inner ZP volume (central cavity within the ZP) were larger and associated with oocytes that failed to develop. These results suggest that administration of eFSH can increase the number of oocytes collected per cycle. Oocyte morphology differed with age and was associated with developmental competence.

Randomized, Controlled, Crossover trial of Prevention of Clindamycin-Induced Gastrointestinal Signs Using a Synbiotic in Healthy Research Cats.

BACKGROUND: Synbiotics often are prescribed to limit antibiotic-associated gastrointestinal signs (AAGS) in cats, but data to support this recommendation are lacking. OBJECTIVE: To determine whether synbiotic co-administration mitigates AAGS in healthy research cats treated with clindamycin. ANIMALS: 16 healthy research cats. METHODS: A randomized, double-blinded, placebo-controlled, 2-way, 2-period, crossover study with a 6-week washout was performed. Each study period consisted of a 1-week baseline and a 3-week treatment period. Cats received 75 mg clindamycin with food once daily for 3 weeks, followed 1 hour later by either 2 capsules of a synbiotic or placebo. Food consumption, vomiting, fecal score, and completion of treatment were compared using repeated measures split plot or crossover designs with covariates, with P < 0.05 considered significant. RESULTS: Cats that received the synbiotic were more likely to complete treatment in period 1 (100% vs. 50%, P = 0.04). Cats vomited less when receiving the synbiotic but this was not significant, but there were significant period effects (F-value = 11.4, P < 0.01). Cats had higher food intake while receiving the synbiotic (F-value = 31.1, P < 0.01) despite period effects (F-value = 8.6, P < 0.01). There was no significant effect of treatment on fecal scores, which significantly increased over time (F-value = 17.9, P < 0.01). CONCLUSIONS AND CLINICAL IMPORTANCE: Administration of a synbiotic 1 hour after clindamycin administration decreased hyporexia and vomiting in healthy cats. Additionally, significant period effects suggest that clinical benefits of synbiotic administration persist for at least 6 weeks after discontinuation, decreasing the severity of AAGS in cats that subsequently received clindamycin with placebo. Unlike in people, synbiotic administration did not decrease antibiotic-associated diarrhea.

Factors affecting the success of oocyte transfer in a clinical program for subfertile mares.

Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2 +/- 0.4 yr of age (mean +/- S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0+/-0.1h after administration of hCG to estrous donors and cultured 16.4 +/- 0.2 h prior to transfer into recipients' oviducts. At 16 and 50 d after transfer, pregnancies were detected in 201 of 504 (40%) and 159 of 504 (32%) of recipients, respectively, with an embryo-loss rate of 21% (42/201). Pregnancy rates were similar (P > 0.05) for cyclic and noncyclic recipients and for recipients inseminated with cooled, fresh or frozen semen. One or more recipients were detected pregnant at 16 and 50 d, respectively, for 80% (69/86) and 71% (61/86) of donors. More donors <20 than > or = 20 yr (mean ages +/- S.E.M. of 15.5 +/- 0.4 and 23.0 +/- 0.3 yr, respectively) tended (P = 0.1) to have one or more pregnant recipients at 50 d (36/45, 80%; 28/45, 62%, respectively). Results of the program confirm that pregnancies can consistently be obtained from older, subfertile mares using oocyte transfer.

Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment.

Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll-centrifuged or accessory sperm cells.

Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol.

Survival of IVF-derived bovine embryos of different ages and stages of development, produced in 2 different co-culture systems and frozen in 2 different cryoprotectants, was investigated. In vitro-derived bovine embryos (n = 5,525) were utilized to study survival following exposure to cryoprotectants and after freezing. Survival of the frozen embryos was based on blastocyst re-expansion 24 h and hatching 72 h after thawing. There was no difference in survival when embryos were exposed to either glycerol (Gly) or ethylene glycol (EG) for 10 or 40 min with the cryoprotectant diluted with or without freezing. In 2 of 3 experiments in which a comparison was possible, more blastocysts frozen in 1.4 M glycerol than in 1.5 M ethylene glycol survived. Addition of 0.25 M sucrose to 1.5 M ethylene glycol in the freezing solution did not improve embryo survival. More blastocysts frozen on Day 7 of in vitro culture survived than those frozen on Day 6 or Day 8. On Days 6, 7 and 8, embryos in the most advanced stage of development survived better than those at less advanced stages. Post-thaw survival did not differ for embryos produced in co-culture with Buffalo Rat Liver (BRL) cells with either Menezo B2 Medium or Tissue Culture Medium 199 and frozen in 1.4 M glycerol.

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts.

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.

The reliability of detecting digital dermatitis in the milking parlour.

Digital dermatitis (DD) is currently the most problematic infectious skin disease in dairy cattle associated with lameness. Reducing the disease prevalence through early detection and treatment is an essential management tool. The traditional detection method involves lifting and inspecting the feet in a cattle crush, but this is a time intensive and costly practice and impractical for regular detection of individual cases or monitoring herd prevalence. This study aimed to establish the accuracy of detecting and classifying DD lesions in traditional (pit) milking parlours compared with a borescope, and a gold standard lifted foot inspection. With the exception of one lesion, parlour screening was as accurate as the lifted foot inspection in determining the presence of 86 DD lesions on 160 hind feet (99% agreement; κ 0.99; sensitivity 1.00; specificity 0.99). Describing lesions by colour, depth or stage of lesion in the parlour or using the borescope reached substantial agreement with the gold standard. The stage of lesion was closely linked to colour and depth descriptors. There was greater agreement when categorising more advanced stages of disease progression. Borescope and parlour inspections led to both over and under recording of actual size, particularly in smaller lesions. Screening cows in traditional milking parlours for the presence of DD was found to be an accurate and practical means of detecting lesions. This method should be considered for on farm use to evaluate DD prevention and treatment strategies.

An investigation into the use of infrared thermography (IRT) as a rapid diagnostic tool for foot lesions in dairy cattle.

Previous research has suggested that temperature at the coronary band increases in the presence of claw horn lesions in dairy cattle. However the reliability of using infrared thermography (IRT) as a method of distinguishing between lesions has not been investigated. The objective of this study was to examine the potential of IRT as a non-invasive tool for rapidly screening for the presence of digital dermatitis (DD). Eighty-two cows which either had no skin lesions on the hind feet (controls, n=41 feet) or a DD lesion on one or both feet were selected during milking. Following selection, each cow was moved into the farm crush where thermal images were taken from the plantar aspect of each foot at the pastern when the foot was dirty, cleaned and raised for visual inspection. Following recording of thermal images each hind foot was trimmed and the presence of any lesion recorded. It was found that the temperature did not differ significantly between feet with DD lesions and other skin or claw horn lesions, regardless of whether the feet were dirty, clean or raised for inspection. As IRT was not sensitive enough for lesion specific detection, the reliability of setting a temperature threshold above which any type of lesion causing lameness could be detected was investigated. The optimal trade off between sensitivity and specificity could be reached without having to either clean or lift the feet. Setting the threshold for disease at 27°C for dirty feet identified 80% of feet with lesions and 73% of feet without lesions correctly. In conclusion, IRT was reliable in detecting elevated temperature associated with foot lesions. Future research investigating the development of lesions may identify a temperature threshold for early treatment intervention. This technique may also be useful to quantify the effectiveness of early treatment by tracking recovery and recurrence of cases.

Reductive pentose cycle and formate assimilation in Rhodopseudomonas palustris.

Rhodopseudomonas palustris assimilated formate autotrophically as carbon dioxide and hydrogen arising from the activity of the formic hydrogenlyase system. Kinetic analyses of cell suspensions pulse-labeled with (14)C-formate or (14)C-bicarbonate showed similar distributions of incorporated radioactivity. In both cases phosphate esters were the first assimilation products. Ribulose diphosphate carboxylase, phosphoribose isomerase, and phosphoribulokinase, characteristic enzymes of the reductive pentose cycle, were present in extracts of cells grown on formate.

Histiocytic ulcerative colitis in three non-boxer dogs.

Histiocytic ulcerative colitis (HUC) is described in three non-boxer dogs. Clinical signs were typical of large-bowel diarrhea and included soft stool, hematochezia, tenesmus, and an increased frequency of defecation. Diagnosis in each case was made by light microscopic evaluation of endoscopically obtained colonic biopsy specimens. Treatment regimes varied, but included immunosuppressive agents, anti-inflammatory drugs, antimicrobials, and dietary modification. Clinical response was substantial in two patients, while the third patient was euthanized due to treatment failure. The authors' observations indicate that HUC may be encountered in non-boxer dogs.

Hematometra secondary to anticoagulant rodenticide toxicity.

An adult, intact female Australian shepherd presented for frank vaginal bleeding of unknown duration. The only coagulation profile abnormality upon presentation was mild prolongation of the partial thromboplastin time (PTT). The uterus was removed at surgery and contained a large amount of coagulated blood. Clotting profiles were markedly abnormal 48 hours postoperatively. Serum analysis was positive for brodifacoum, an anticoagulant rodenticide. Preoperative coagulation was most likely normalized by vitamin K1 therapy administered prior to presentation. The only manifestation of anticoagulant rodenticide was hematometra. Rodenticide intoxication should be considered in the differential diagnosis list of hematometra or metrorrhagia.