Xenogenous fertilization of equine oocytes following recovery from slaughterhouse ovaries and in vitro maturation.

The in vitro production (IVP) of equine embryos using currently available protocols has met limited success; therefore investigations into alternative approaches to IVP are justified. The objective of this study was to evaluate the feasibility of xenogenous fertilization and early embryo development of in vitro matured (IVM) equine oocytes. Follicular aspirations followed by slicing of ovarian tissue were performed on 202 equine ovaries obtained from an abattoir. A total of 667 oocytes (3.3 per ovary) were recovered from 1023 follicles (recovery rate, 65%). Oocytes underwent IVM for 41 +/- 2 h (mean +/- S.D.), before being subjected to xenogenous gamete intrafallopian transfer (XGIFT). An average of 13 +/- 0.8 oocytes and 40x10(3) spermatozoa per oocyte were transferred into 20 oviducts of ewes. Fourteen percent of transferred oocytes (36/259) were recovered between 2 and 7 days post-XGIFT and 36% of those recovered displayed embryonic development ranging from the 2-cell to the blastocyst stage. Fertilization following XGIFT was also demonstrated by the detection of zinc finger protein Y (ZFY) loci. Ligation of the uterotubal junction (UTJ), ovarian structures, or the duration of oviductal incubation did not significantly affect the frequency of embryonic development or recovery of oocytes/embryos after XGIFT. In conclusion, equine embryos can be produced in a smaller non-equine species that is easier for handling.

Effect of platelet-activating factor (PAF) on stallion sperm motility, capacitation and the acrosome reaction.

Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=1-O-alkyl-acetyl-sn-glycero-3-phosphocholine) is a signalling phospholipid that has many biological properties in addition to platelet activation. PAF receptors have been detected on stallion spermatozoa; therefore, the aim of this study was to evaluate the effect of synthetic PAF on the motility, capacitation and the acrosome reaction of stallion spermatozoa. Treatment of ten stallion semen samples with 10(-4)-10(-13) mol PAF l(-1) resulted in significant differences in motility and capacitation (r(2)=0.81 and 0.83, respectively). Statistical analysis indicated that PAF also has an effect on acrosome reaction (r(2)=0.20). PAF concentrations, incubation time and their interaction had a highly significant (P<0.01) effect on motility. After capacitation in vitro with PAF, and induction of the acrosome reaction by progesterone, transmission electron microscopy was conducted on the spermatozoa of three stallions to detect the true acrosome reaction. Differences in PAF concentrations were highly significant (r(2) for intact: 97.2; reacted: 89.8; and vesiculated: 98.1). The results indicate that a lower concentration of PAF enhances motility and induces capacitation of stallion spermatozoa, whereas a higher concentration of PAF induces the acrosome reaction.

Interpretation of the detection of Sarcocystis neurona antibodies in the serum of young horses.

Horses that are exposed to Sarcocystis neurona, a causative agent of equine protozoal myeloencephalitis, produce antibodies that are detectable in serum by western blot (WB). A positive test is indicative of exposure to the organism. Positive tests in young horses can be complicated by the presence of maternal antibodies. Passive transfer of maternal antibodies to S. neurona from seropositive mares to their foals was evaluated. Foals were sampled at birth (presuckle), at 24h of age (postsuckle), and at monthly intervals. All foals sampled before suckling were seronegative. Thirty-three foals from 33 seropositive mares became seropositive with colostrum ingestion at 24h of age, confirming that passive transfer of S. neurona maternal antibodies occurs. Thirty-one of the 33 foals became seronegative by 9 months of age, with a mean seronegative conversion time of 4.2 months. These results indicate that evaluation of exposure to S. neurona by WB analysis of serum may be misleading in young horses.

In vitro and xenogenous capacitation-like changes of fresh, cooled, and cryopreserved stallion sperm as assessed by a chlortetracycline stain.

Like the human female, the mare experiences reproductive tract pathology that may sometimes be circumvented by the use of assisted reproductive technologies (ARTs). One such technology, gamete intrafallopian transfer (GIFT), may be used in mares that exhibit ovulatory, oviductal, or uterine abnormalities that limit the use of common ARTs, such as embryo transfer. Homologous GIFT has been successfully performed in the horse; however, the logistics, costs, and associated risks of surgically transferring gametes to the oviducts of a recipient mare are considerably high. Use of a less costly species in a heterologous or xenogenous procedure would therefore be beneficial. This study represents the preliminary investigation into the use of sheep as recipients for xenogenous GIFT procedures using equine gametes. We investigated the capacitation response of fresh, cooled, or frozen stallion sperm after 1) in vivo incubation in the reproductive tract of estrous and anestrous ewes as well as 2) in vitro incubation in a modified Krebs/ Ringer extender at 37 degreesC with and without the addition of heparin at 10 IU/mL for up to 8 hours. A chlortetracycline (CTC) fluorescent stain was used to assess the capacitation response of sperm. Findings indicated that oviductal fluid samples recovered from estrous ewes had significantly higher numbers of sperm exhibiting capacitation-like staining patterns when compared to samples recovered from anestrous ewes (P < .05). Fresh semen yielded higher capacitation-like staining patterns after in vivo incubation than did frozen-thawed or cooled samples. A transition from majority CTC unreacted sperm to majority CTC non-acrosome intact sperm was demonstrated for both in vivo and in vitro studies. In vitro incubation of stallion sperm with heparin did not result in an increased capacitation-like staining response over time when compared with nonheparinized samples. Results from this study suggest that xenogenous capacitation of stallion sperm may occur in the estrous ewe.

Dietary carbohydrates and fat influence radiographic bone mineral content of growing foals.

Hydrolyzable carbohydrate intake in horse diets may become excessive when rapidly growing pastures are supplemented with grain-based concentrates. The substitution of fat and fiber for hydrolyzable carbohydrate in concentrates has been explored in exercising horses but not in young, growing horses. Our objective was to compare bone development in foals that were fed pasture and concentrates rich in sugar and starch (corn, molasses) or fat and fiber (corn oil, beet pulp, soybean hulls, oat straw). Forty foals were examined, 20 each in 1994 and 1995. In each year, 10 mares and their foals were fed a corn and molasses supplement (SS) and 10 others were fed a corn oil and fiber supplement (FF). The concentrates were formulated to be isocaloric and isonitrogenous, and mineral content was balanced to complement the pastures and meet or exceed NRC requirements. Dorsopalmar radiographs were taken of the left third metacarpal monthly from birth to weaning and then every other month until 1 yr of age. Bone density was estimated using imaging software and an aluminum stepwedge. Radiographic examination indicated differences in medial, lateral, and central bone mineral content of the metacarpal III. Bone mineral content increased with age, and a plateau was observed during winter. Bone mineral content was lower in weanlings and yearlings fed the FF supplement than in those fed SS. Subjective clinical leg evaluations indicated differences in physitis, joint effusion, and angular and flexural limb deformities in response to age, and possibly to season. Regression analysis indicated positive relationships between bone mineral content and body weight, age, and body measurements. Nutrient and chemical interactions, such as the binding of calcium by fat and fiber, may alter the availability of elements necessary for bone development.

Effect of phenylbutazone and flunixin meglumine on acute toxic mastitis in dairy cows.

A double-blinded randomized prospective clinical trial was designed to evaluate the effectiveness of flunixin meglumine and phenylbutazone for treatment of acute toxic mastitis in dairy cows. All cows were treated 4 times at 12-hour intervals by intramammary infusion of gentamicin (150 mg). A total of 45 dairy cows with toxic mastitis were randomly assigned to 1 of 3 treatment groups: group 1 (control), saline solution, IV; group 2, 1 g of flunixin meglumine, IV; or group 3, 4 g of phenylbutazone, IV. Physical examination and udder variables were assessed at initial examination and 24 hours later. Milk production was recorded at regular intervals from 1 week before until 10 weeks after development of mastitis. Rear quarters (34/45) were more commonly affected than front quarters. Thirty-five cows returned to the herd, 9 cows were culled, and 1 cow died. There were no significant differences among treatment groups in the need for further treatment or outcome. Klebsiella spp (18/45) and Escherichia coli (16/45) were the most common pathogens isolated by culture of milk from affected quarters. The overall bacteriologic cure rate on days 7 and 14 was 64 and 75%, respectively. At the time of initial examination, cows of the control group had higher rectal temperature than did cows of the flunixin group. At the examination 24 hours later, the rectal temperature of cows in all treatment groups was lower than the temperature at initial examination; at that time (24 hours), however, there were no significant differences in temperature among the treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Uterine tear without a corresponding placental lesion in a mare.

A 3-cm full-thickness tear near the tip of the previously gravid uterine horn of a 12-year-old Thoroughbred mare was diagnosed at surgery. The mare had delivered a foal with veterinary assistance 2 days prior to surgery. The mare subsequently developed muscle tremors, anorexia, and signs of depression. Physical examination revealed pyrexia, extended capillary refill time, muscle fasciculations, and fewer-than-normal borborygmi. Abnormalities of the uterus or vagina were not detected by palpation per rectum or per vagina. The chorioallantois underlying the area of the tear was intact. We propose that the uterine tear was caused by pressure created by the foal's stifle against the maternal pelvis or by blunt forces caused by the foal's foot, sufficient to tear the uterus, but not the chorioallantois.

Differentiation of chronic lymphocytic leukemia in the horse. A report of two cases.

Chronic lymphocytic leukemia (CLL) was diagnosed in two horses: an 18-year-old Quarter Horse gelding that was examined because of edema of the prepuce and ventral abdomen; and a 20-year-old mixed breed gelding that was referred because of lymphocytosis, ventral edema, and weight loss. The first horse had enlarged peripheral lymph nodes and cool nonpainful pitting edema of the ventral abdomen and prepuce. The second horse had enlarged peripheral lymph nodes, cool nonpainful pitting edema of the ventral thorax and cranial ventral abdomen, and a 3/5 holosystolic heart murmur. The diagnosis of CLL was based on increased blood lymphocyte counts and infiltration of marrow and other tissues by lymphocytes. In horse 1, the lymphocytosis persisted for 2 months between initial examination and death. The results of flow cytometric analysis on blood lymphocytes using anti-lymphocyte antibodies suggested that horse 1 had T-cell CLL, and horse 2 had B-cell CLL. In addition, the second horse had a monoclonal gammopathy (IgG), with light-chain proteinuria.

A case-control study of the association between blood selenium and cystic ovaries in lactating dairy cattle.

We carried out a case-control study to investigate the association between the risk of developing cystic ovaries and the concentrations of Se in the blood of dairy cows. Cows were from dairy herds in the vicinity of Ithaca, NY. One hundred and thirty-two cystic ovary cases were identified; a similar number of herdmates who did not have cystic ovaries were selected as controls. Whole blood samples were collected from cystic cows and controls in which Se concentrations were determined. Milk samples also were collected and analyzed for concentrations of progesterone. Multivariate logistic regression analysis was used to identify the role of Se in the risk of developing cystic ovaries while controlling for other possible risk factors. Mean Se in blood concentration for cows with cystic ovaries (141 ng/ml) was similar to that of control cows without cystic ovaries (136 ng/ml). The concentration of progesterone in milk was significantly higher in controls than in cows with cystic ovaries. In the multivariate logistic regression analysis, cows with concentrations of Se in blood greater than 169 ng/ml had twice the risk of developing cystic ovaries compared with cows that had Se levels less than 108 ng/ml.