Comparison of ram sperm interaction with bovine cervical mucus

Ram sperm penetration in estrous bovine cervical mucus was evaluated from ejaculates collected during long (16L:8D) and short (8L:16D) photoperiods with varying ambient temperatures. The distance traveled by vanguard sperm was affected by an interaction of the photoperiod and temperature (P<0.001). Sperm migration distance in a capillary tube filled with mucus (22.5 to 23.2 mm) was greater when sperm were collected from rams on short days and when the ambient temperature was between 10 and 31 degrees C than when sperm were collected under either long or short days (15.5 to 17.8 mm), when ambient temperatures were between 1 to 9 degrees C. Incidence of head-to-head agglutination of sperm differed by temperature (P<0.05) and photoperiodic (P<0.09) conditions. The percentage of ejaculates with evidence of sperm agglutination in the mucus was higher in long (62.5%) vs short (45.2) days, and it was greater in sperm collected in warm (61%) vs cold (44%) days. Physical interaction of cervical mucus with spermatozoa was examined. The binding of an iodinated protein from lyophilized mucus to a detergent soluble extract of washed or unwashed sperm was observed. These data show that both photoperiod and temperature affect the interaction of ram sperm with bovine cervical mucus.     

Fertilization and embryo loss in Booroola Merino x South Australian Merino ewes: Effect of the F gene

The effects of Booroola genotype (F+, ++); the number of ovulations per ewe (one, two or three); and the age of a ewe (2.5 yr vs 3.5 to 6.5 yr) on the percentage of ova fertilized, embryo loss and fetal loss were examined in Booroola x South Australian Merino ewes slaughtered on Days 4, 21 and 90 after insemination. Ewes slaughtered on Day 90 were examined by real-time ultrasound imaging (RUI) on Day 45. Fertilization failure was independent of ewe genotype, ovulation rate and age of ewe, and it was not an important source of wastage (F+, 9.4%; ++, 6.7%). Most embryo loss occurred during the first 21 d (F+, 54.7%; ++, 40.3%). Interpretation of the effects of genotype and ovulation rate on embryo wastage measured on Days 21, 45 and 90 was obscured by significant (P < 0.05) genotype and ovulation rate interactions with the day of slaughter/RUI. The effect of age on embryo loss was not significant (P > 0.05). Reasons for the high rate of wastage observed in this experiment require further study.     

Effects of naloxone and animal temperament on serum luteinizing hormone and cortisol concentrations in seasonally anestrous Brahman heifers

The effect of endogenous opioid peptides (EOP) and individual animal temperament on serum luteinizing hormone (LH) were investigated in seasonally anestrous Brahman heifers (n = 24). Animals that had shown behavioral estrus in previous months but that had not returned to estrus for at least 30 d were selected. The heifers were ranked by temperament (tame = 1, normal = 2, wild = 3) and randomly allotted into three groups. Blood was collected from one heifer of each group per day. Blood samples were taken via jugular cannula every 15 min for 6 h and every 30 min for another 4 h. After the first hour of sampling, the heifers received intravenous saline (SAL, n = 8); naloxone (LN, 0.5 mg/kg i.v., n = 8); or naloxone (HN, 1.0 mg/kg i.v., n = 8). Three hours after naloxone treatment, each heifer was given gonadotropin releasing hormone (GnRH, 100 mug i.m.). All samples were processed to yield serum and were assayed for LH by radioimmunoassay (RIA). Hourly samples were assayed for cortisol by RIA. The area under the LH curve 60 min postnaloxone treatment was higher in LN and HN than in SAL (57.0 and 40.8 vs 6.1 units; P<0.01); and the area under the 180 min postnaloxone curve remained higher in LN than in SAL (106.2 vs 35.1 units; P<0.05). Cortisol concentrations 60 min postnaloxone administration were above prenaloxone levels(38.2 vs 26.7 ng/ml; P<0.0002). Temperament scores of heifers were positively correlated with cortisol release. The area under the cortisol curve had a negative correlation with mean LH. Serum LH concentrations appear to be suppressed by EOP in seasonally anestrous Brahman heifers, and EOP appear to reduce serum cortisol concentrations. Excitable heifers had higher concentrations of serum cortisol, which negatively affected serum LH concentrations.     

Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39 degrees C under 5% CO(2) in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.     

A technique for transcervical intrauterine insemination of ewes

In commercial artificial insemination (AI) of sheep, fresh extended semen is deposited into the vagina or cervical os, or fresh extended or frozen semen is placed laparoscopically into the uterus. Transcervical intrauterine insemination of the ewe is not used commercially. In this study, methods of restraint and instrumentation for AI were evaluated and modified to produce a transcervical intrauterine technique suitable for commercial application. Four methods of restraint, four vaginal specula, three forceps and four instruments suitable for transcervical passage were compared. From these comparisons a technique was developed in which the ewes were positioned in dorsal recumbency with their hindquarters elevated. The vagina was dilated using a duck-billed speculum, the cervix was grasped and retracted using forceps, and an inseminating instrument was introduced into the cervical opening and manipulated through the cervical canal. The technique was repeated on 89 mature, multiparous ewes: the difficulty in locating the cervical opening, the force required to retract the cervix and the time required to penetrate into the uterus were recorded. Uterine penetration was achieved in 82% of the ewes. This technique has the potential to be applied in commercial artificial insemination programs of sheep.     

Sequential uterine horn versus simultaneous total uterine flush to recover bovine embryos nonsurgically

Beef cows were superovulated with follicle stimulating hormone (FSH) to compare two nonsurgical methods of embryo recovery from the uterus. In the first method each uterine horn was independently flushed with physiological saline solution (PSS) through a Foley catheter passed through the cervix and into the uterine horn. In the second method both uterine horns were simultaneously flushed with PSS by passing the catheter into the uterine body. In both methods, the numbers of ovulations were determined after embryo collection by counting the corpora lutea (CL) on both ovaries of each cow through a flank incision. Independent flushing (n = 19) averaged 6.4 embryos and 16.1 CL per cow for a recovery rate of 40%. Simultaneous flushings (n = 22) averaged 5.4 embryos and 17.7 CL per cow for a recovery rate of 31%. This difference between the recovery rates of the two flushing methods was not significant (P>0.05).     

Selection of Astaxanthin-Overproducing Mutants of Phaffia rhodozyma with beta-Ionone

beta-Ionone, an end ring analog of beta-carotene, inhibits astaxanthin production in the red yeast Phaffia rhodozyma. Astaxanthin-overproducing mutants of this yeast are easily spotted on beta-ionone-containing yeast malt agar plates. beta-Ionone appears to block astaxanthin synthesis at the beta-carotene level.     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H(2)-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H(2)-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 +/- 0.19 mol%), a phospholipid pattern of type II, MK-9(H(4)) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t(d) = 8 h), H(2) plus CO(2) (t(d) = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H(2), nickel was not necessary. The optimum growth temperature was 65 degrees C; no growth was observed below 40 degrees C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70 degrees C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K(m) was 88 mul of CO liter and V(max) was 20.2 mul of CO h mg of protein. The threshold value of S. thermoautotrophicus of 0.2 mul of CO liter was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 mumol min mg of protein. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.