Estimated impact of multiple conceptuses per follicle on fecundity in the bitch.

Multiple conceptuses from one follicle due to multi-ovular follicles or monozygotic twins inflate the ratio between the numbers of conceptuses and corpora lutea and possibly the effect ascribed to experimental treatment. The aim of this study was to estimate the impact of multiple conceptuses per follicle on fecundity in the bitch. The numbers of conceptuses and corpora lutea of 105 bitches originated from 7 published experimental studies and of 132 from ovariohysterectomies done in a private practice and a welfare organisation. Ninety five bitches, producing 597 conceptuses, had at least as many conceptuses as corpora lutea; 83 had as many, 10 had one more and 2 had 2 more. Bootstrap resampling on these 95 bitches let us to conclude that 12.6% of bitches are expected to produce more oocytes that are each capable of yielding a conceptus than the number of follicles that ovulate and that 2.35% of conceptuses are expected to be in excess of the number of follicles that ovulate. Applying the results of the current study to 2 earlier studies showed that multiple conceptuses from one follicle may, at maximum, have increased the effect ascribed to prostatic fluid by 0.022 and 0.024 conceptuses per corpus luteum, which is minor in relation to the magnitude of the ascribed effects of 0.34 and 0.22 conceptuses per corpus luteum. The number of conceptuses relative to the number of corpora lutea provides a valid and precise measurement of fertility in the bitch.

Validation of merocyanine 540 staining as a technique for assessing capacitation-related membrane destabilization of fresh dog sperm.

The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that had undergone membrane stabilization known to be associated with capacitation in other species, and whether such destabilization is detected earlier than when using the tyrosine phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence microscopy. Semen from nine dogs was collected and incubated in parallel in bicarbonate-free modified Tyrode's medium (-BIC), medium containing 15 mM bicarbonate (+BIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6 hours. Staining with M540-YP allowed the classification of dog sperm as viable without destabilized membranes, viable with destabilized membranes, nonviable without destabilized membranes, or nonviable with destabilized membranes. The percentage of viable sperm detected using EH (83.5 ± 1.37%; mean ± SEM) was higher than when using YP (66.7 ± 1.37%: P < 0.05; n = 54 semen samples). On the other hand, M540-YP identified a higher percentage of viable sperm with destabilized membranes than TP-EH (75 ± 1.76% vs. 35 ± 1.70%: P < 0.05; n = 54 semen samples). Staining with M540-YP indicated a rapid increase in the percentage of viable sperm with destabilized membranes, reaching a maximum during the first 30 minutes of incubation in +BIC. For all other treatments (i.e., -BIC, prostatic fluid, and PBS), the peak in the percentage of viable sperm with destabilized membranes was reached as much as 90 to 210 minutes later than incubation in +BIC. The lowest percentage of viable sperm showing signs of capacitation was recorded during incubation in PBS. We conclude that YP identifies sperm committed to cell death earlier than EH, and that the M540-YP stain combination identifies membrane destabilization known to be associated with capacitation in other species earlier than the TP-EH stain combination.

Prediction of the optimal time for insemination using frozen-thawed semen in a multi-sire insemination trial in bitches.

The aims of the study were to determine which of Days 5, 6 or 7 after the blood plasma progesterone concentration (PPC) of bitches first reached 6-9 nmol/L (Day 0) yield the highest fertility and whether day of insemination affects the gender ratio of conceptuses. Six bitches were inseminated on Days 5 and 6 and 6 on Days 6 and 7. Ten million progressively motile frozen-thawed sperm from each of 5 dogs were pooled for the first insemination. The same number of sperm from 5 other dogs were pooled for the second insemination. Only one batch of semen from each dog was used on all bitches, which largely prevented any effect of male and semen. Twenty-three autosomal microsatellites and the amelogenin gene were used to determine the paternity and gender of the conceptuses. Pregnancy rate was 100%. Out of 103 ovulations 66 conceptuses were conceived (conception rate: 64%). The proportion of available oocytes fertilised was 0.11, 0.56, and 0.27 for Days 5, 6, and 7, respectively. The odds of fertilisation was 16.7 and 4.2 times higher from insemination on Day 6 compared to Day 5 (P<0.001) and Day 7 (P = 0.013), respectively. The numbers of male- and female conceptuses were equal (33 each) and gender was independent of insemination day (P = 0.18). This study suggests that intrauterine insemination of bitches should best be done 6 days after PPC first reaches a value between 6 and 9 nmol/L with a second insemination one day later.

Ex vivo influence of carbetocin on equine myometrial muscles and comparison with oxytocin.

To determine the intercyclic effect of oxytocin and carbetocin on equine myometrial tissue, the effect of the drugs was evaluated through pharmacokinetic and pharmacodynamic studies. The complete pharmacokinetic profile for oxytocin was unknown and had to be established. To do so, 25 IU of oxytocin were administered intravenously to six cycling mares and blood samples were collected before and 2, 4, 8, and 15 min after administration. The half-life of oxytocin was determined to be 5.89 min, the clearance rate 11.67 L/min, mean residence time (MRT) 7.78 min. The effective plasma concentration was estimated to be 0.25 ng/mL. This was similar to the concentration achieved for the organ bath study where the concentration that produced 50% of the maximum effect (EC(50)) was calculated at 0.45 ng/mL. To determine the intercyclic effect of oxytocin and carbetocin uterine myometrial samples were collected from slaughtered mares in estrus, diestrus, and anestrus. The samples were mounted in organ baths and exposed to four ascending, cumulative doses of oxytocin and carbetocin. Area under the curve and amplitude, maximum response (E(max)), and concentration that produced 50% of the maximum effect were studied for each agonist and statistically evaluated. The effect of oxytocin on equine myometrial tissue was higher during diestrus, and surprisingly anestrus, than during estrus, whereas the effect of carbetocin was the same independent of the stage of estrous cycle. A significant difference was found for estrous and anestrous samples when oxytocin was used but not when carbetocin was used.

Seroprevalence of canine herpesvirus in breeding kennels in the Gauteng Province of South Africa.

Canine herpesvirus (CHV-1) causes neonatal deaths as well as infertility due to embryonal death, abortion and stillbirths in breeding kennels. The aim of this study was to determine the prevalence of antibodies against canine herpesvirus in the serum of dogs older than 1 year in breeding kennels in the Gauteng Province of South Africa. A serum neutralization test (SNT) and a newly developed enzyme linked immunosorbent assay (ELISA) were used to test the serum samples of 328 dogs in 38 breeding kennels. With SNT as well as ELISA, 22% of sera were positive (P>0.9). Seventeen kennels (45% of total kennels) each had at least one positive dog on SNT compared with twenty kennels (53% of total kennels) that each had at least one positive dog on ELISA (P=0.6). The prevalence of positive dogs in positive kennels was 42+/-26% (n=17 kennels) for SNT and 39+/-26% (n=20 kennels) for ELISA. Pairwise comparison of kennels showed that the prevalence of SNT positive dogs was similar to the prevalence of ELISA positive dogs (P=0.3, n=38 kennels). Seroprevalence was independent of age, gender or colony size. This study suggests that canine herpesvirus is sufficiently common in breeding dogs in the Gauteng Province of South Africa to pose a threat to neonatal survival and fertility.

Characterization of three distinct cDNA clones encoding cysteine proteinases from maize (Zea mays L.) callus.

In previous work, a 33 kDa cysteine proteinase was found in callus initiated from maize (Zea mays L.) resistant to fall armyworm feeding. A callus cDNA library from the maize inbred Mp708 was screened with oligonucleotides derived from the N-terminal amino acid sequence of the 33 kDa proteinase and several cDNA clones were isolated and sequenced. A cDNA clone encoding the 33 kDa cysteine proteinase, mir1, was identified. Two additional clones, mir2 and mir3, encoding putative cysteine proteinases were also identified. mir2 and mir3 are distinct from mir1 and each other, but show a high degree of homology. All of the mir cDNA clones map to distinct sites on the maize genome. Amino acid sequences encoded by the mir clones are similar to other known cysteine proteinases and are most closely related to the oryzain-alpha and -beta precursors. The ERFNIN motif and a 12 amino acid conserved sequence are present in the propeptide region of the putative proteinases encoded by mir clones. mir2 and mir3 appear to have C-terminal extensions. The phylogenetic tree of nucleotide sequences of mir1, mir2, mir3 and other representative cysteine proteinases from protozoa, plants and animals was constructed.

Tissue banking: the role of the Regional Blood Centre: an American experience in Minnesota.

The clinical use of bone, cornea, skin, connective tissue and heart valve allografts has increased greatly in the past several years, with bone the most commonly transplanted tissue. Tissue allografts have been the vehicle to transmit infectious disease from donor to recipient. Provision of safe and effective tissue has gradually developed and improved as regional tissue banks have organized, and standards of practice evolved. Regional Blood Centres have the special capabilities and experience to provide the expertise required in tissue banking. Consequent to these community needs, regional bone and tissue banks have been established in blood banks and the experience of the American Red Cross in St Paul, Minnesota is described.

Thermodynamics of isomerization reactions involving sugar phosphates.

Thermodynamics of isomerization reactions involving sugar phosphates have been studied using heat-conduction microcalorimetry. For the process glucose 6-phosphate2-(aqueous) = fructose 6-phosphate2- (aqueous), K = 0.285 +/- 0.004, delta Go = 3.11 +/- 0.04 kJ.mol-1, delta Ho = 11.7 +/- 0.2 kJ.mol-1, and delta Cop = 44 +/- 11 J.mol-1.K-1 at 298.15 K. For the process mannose 6-phosphate2- (aqueous) = fructose 6-phosphate2- (aqueous), K = 0.99 +/- 0.05, delta Go = 0.025 +/- 0.13 kJ.mol-1, delta Ho = 8.46 +/- 0.2 kJ.mol-1, and delta Cop = 38 +/- 25 J.mol-1.K-1 at 298.15 K. The standard state is the hypothetical ideal solution of unit molality. An approximate result (-14 +/- 5 kJ.mol-1) was obtained for the enthalpy of isomerization of ribulose 5-phosphate (aqueous) to ribose 5-phosphate (aqueous). The data from the literature on isomerization reactions involving sugar phosphates have been summarized, adjusted to a common reference state, and examined for trends and relationships to each other and to other thermodynamic measurements. Estimates are made for thermochemical parameters to predict the state of equilibrium of the several isomerizations considered herein.

Thermodynamics of hydrolysis of sugar phosphates.

Thermodynamics of the enzyme-catalyzed (alkaline phosphatase, EC 3.1.3.1) hydrolysis of glucose 6-phosphate, mannose 6-phosphate, fructose 6-phosphate, ribose 5-phosphate, and ribulose 5-phosphate have been investigated using microcalorimetry and, for the hydrolysis of fructose 6-phosphate, chemical equilibrium measurements. Results of these measurements for the processes sugar phosphate2- (aqueous) + H2O (liquid) = sugar (aqueous) + HPO2++-(4) (aqueous) at 25 degrees C follow: delta Ho = 0.91 +/- 0.35 kJ.mol-1 and delta Cop = -48 +/- 18 J.mol-1.K-1 for glucose 6-phosphate; delta Ho = 1.40 +/- 0.31 kJ.mol-1 and delta Cop = -46 +/- 11 J.mol-1.dK-1 for mannose 6-phosphate; delta Go = -13.70 +/- 0.28 kJ.mol-1, delta Ho = -7.61 +/- 0.68 kJ.mol-1, and delta Cop = -28 +/- 42 J.mol-1.K-1 for fructose 6-phosphate; delta Ho = -5.69 +/- 0.52 kJ.mol-1 and delta Cop = -63 +/- 37 J.mol-1.K-1 for ribose 5-phosphate; and delta Ho = -12.43 +/- 0.45 kJ.mol-1 and delta Cop = -84 +/- 30 J.mol-1.K-1 for the hydrolysis of ribulose 5-phosphate. The standard state is the hypothetical ideal solution of unit molality. Estimates are made for the equilibrium constants for the hydrolysis of ribose and ribulose 5-phosphates. The effects of pH, magnesium ion concentration, and ionic strength on the thermodynamics of these reactions are considered.

Thermodynamics of the hydrolysis of adenosine 5'-triphosphate to adenosine 5'-diphosphate.

The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.

Thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia.

The thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia have been investigated using both heat conduction microcalorimetry and high-pressure liquid chromatography. The reaction was carried out in aqueous phosphate buffer over the pH range 7.25-7.43, the temperature range 13-43 degrees C, and at ionic strengths varying from 0.066 to 0.366 mol kg(-1). The following values have been found for the conversion of aqueous L-aspartateH- to fumarate2- and NH4+ at 25 degrees C and at zero ionic strength: K = (1.48 +/- 0.10) x 10(-3), DeltaG degrees = 16.15 +/- 0.16 kJ mol(-1), DeltaH degrees = 24.5 +/- 1.0 kJ mol(-1), and DeltaC(p) degrees = -147 +/- 100 J mol(-1) K(-1). Calculations have also been performed which give values of the apparent equilibrium constant for the conversion of L-aspartic acid to fumaric acid and ammonia as a function of temperature, pH and ionic strength.

High Precision Microcalorimetry: Apparatus, Procedures, and Biochemical Applications.

Apparatus and procedures used for high-precision microcalorimetric measurements are described. The calorimeter is of the heat-conduction type and utilizes semi-conductor thermoelectric modules. The bicompartmental reaction vessel is made of high-density polyethylene and holds about 0.5 mL of solution in each compartment. Imprecision of heat measurement is 0.2 percent when measuring 300 mJ of heat produced by a rapid chemical reaction. Three microcalorimeters are operated simultaneously using a microcomputer and a data acquisition system. Thermochemical and kinetic applications are described. The acquisition of data from an isoperibol solution calorimeter is also described.

Thermodynamics of the conversion of fumarate to L-(-)-malate.

The thermodynamics of the conversion of aqueous fumarate to L-(-)-malate has been investigated using both heat conduction microcalorimetry and a gas chromatographic method for determining equilibrium constants. The reaction was carried out in aqueous Tris-HCl buffer over the pH range 6.3-8.0, the temperature range 25-47 degrees C, and at ionic strengths varying from 0.0005 to 0.62 mol kg-1. Measured enthalpies and equilibrium ratios have been adjusted to zero ionic strength and corrected for ionization effects to obtain the following standard state values for the conversion of aqueous fumarate 2- to malate 2- at 25 degrees C: K = 4.20 +/- 0.05, delta G degrees = -3557 +/- 30 J mol-1, delta H degrees = -15670 +/- 150 J mol-1, and delta C degrees p = -36 +/- J mol-1 K-1. Equations are given which allow one to calculate the combined effects of pH and temperature on equilibrium constants and enthalpies of this reaction.

Thermodynamics of the conversion of aqueous xylose to xylulose.

The thermodynamics of the conversion of aqueous xylose to xylulose has been investigated using high-pressure liquid chromatography (HPLC) and microcalorimetry. The reaction was carried out in aqueous phosphate buffer over the pH range 6.8-7.4 using solubilized glucose isomerase with MgSO(4) as a cofactor. The temperature range over which this reaction was investigated was 298.15-342.15 K. A combined analysis of both the HPLC and microcalorimetric data leads to the following results at 298.15 K for the conversion process: DeltaG degrees = 4389 +/- 31 J mol(-1), DeltaH degrees = 16090 +/- 670 J mol(-1), and DeltaC(p) degrees = 40 +/- 23 J mol(-1) K(-1). The temperature dependence of the equilibrium constant for the reaction is expressed as R ln K = -4389/298.15 +16090[(1/298.15)-(1/T)]+40[(298.15/T)-1 + ln(T/298.15)]. Comparisons are made with literature data.