S.M. Hopkins

Assessment of boar sperm viability using a combination of two fluorophores

A combination of the fluorophore probes, calcein acetylmethyl ester (CAM) and ethidium homodimer (EH), were used to assess viability of ejaculated boar spermatozoa. Both CAM and EH have been used as indicators of biosynthetic activity and membrane integrity in monolayer cell cultures, with CAM shown to permeate and undergo enzymatic cleavage in viable monolayer cells giving the cell a green fluorescence, and EH penetrating only membrane damaged cells giving cells a red fluorescence. To determine if these fluorophores can be used to assess boar sperm viability, ejaculates from 10 boars were divided into 3 test groups (cytotoxic-treated, swim-up and washed), utilizing a split-ejaculate technique; each group consisted of both a probe-treated and control sample. Sample viability was ascertained in the control groups by visual estimation of the percentage motile spermatozoa, whereas the number of spermatozoa showing green (CAM = viable) or red (EH = non-viable) fluorescence were quantitated for each of the probe-treated groups using a fluorsecein or rhodamine filter, respectively. All spermatozoa exposed to the combined probes had an uptake of one or both fluorophores. The cytotoxic-treated group exhibited 0% gross motility, with 100% of the sperm heads showing red fluorescence. In the swim-up group, no difference was detected (P > 0.05) between control gross motility and the percentage of completely green fluorescing spermatozoa (85% vs. 86.6%, respectively). In the washed group, a significant difference (P = 0.039) was detected between gross motility estimates and the percentage of calcein-green fluorescent spermatozoa (57% vs. 60%, respectively). This study demonstrated that 1) CAM fluoresces only viable sperm, giving off a green fluorescence, 2) EH fluoresces in only non-viable sperm, giving off a red fluorescence, 3) visual estimation of motile sperm can approximate a semen samples viability, but is not as precise as fluorophore determination, and 4) sperm incubation with the fluorophore combination CAM and EH provided an accurate technique for the objective assessment of boar sperm viability via their distinct fluorescent patterns in boar sperm.

Effects of epidural administration of xylazine or lidocaine on bovine uterine motility and perineal analgesia.

The objective of this study was to evaluate and compare the effects of caudal epidural (sacral-coccygeal interspace) administration of xylazine or lidocaine on uterine motility and perineal analgesia in the cow. Six Holstein cows (7 d post estrus) were assigned to one of three treatment groups: control (5 ml saline); lidocaine (0.2 mg/kg, 2% solution); and xylazine (0.06 mg/kg suspended in 5 ml saline), with each cow randomly assigned to each treatment over a period of three estrous cycles. Uterine motility, perineal analgesia, electrocardiography, and overt signs of sedation were recorded. Data were collected at 10-min intervals starting 10 min before treatment and continuing until 60 min post treatment. At 60 min post treatment, oxytocin (20 USP units) was administered i.v. to serve as a positive control for uterine motility. In the xylazine group, uterine motility significantly (P < 0.05) increased at 20 min post treatment, peaked at 30 min, and gradually decreased to non-significant levels at 50 min post treatment when compared with the lidocaine and control groups. Additionally, xylazine produced a higher degree and longer duration of perineal analgesia than lidocaine. Systemically, epidural xylazine produced signs of sedation, salivation, vocalization and bradycardia. Ataxia was also observed in the xylazine-treated group which may have been induced through a local and/or systemic effect. The individual properties of xylazine and lidocaine should be taken into consideration when performing an obstetrical procedure requiring the use of an epidural analgesic agent, and they should be utilized to benefit the clinician in performing the procedure.

Vaginal conductivity as an indicator for optimum breeding time in the sow after weaning

Abstract One hundred crossbreed multiparous sows were probed for vaginal mucosal conductivity using a conductivity meter (OVU-MATETM) to detect optimum time for breeding. The vaginal mucosal conductivity was measured once daily, starting the day of weaning until the sows were bred. According to the manufacturer, a 15 point increase in the OVU-MATE reading, when compared to that of the previous day, indicates the approach of estrus. After weaning the one hundred sows were randomly assigned to one (n = 20) of the five groups according to the following breeding schedules: sows were bred once at 0 h (Group 1), at 12 h (Group 2), at 24 h (Group 3) and at 36 h (Group 4) following a 15 point or higher increase in vaginal mucosal conductivity. Control sows (Group 5) were bred twice, 24 h apart, when estrus was detected by intact boars. All sows were bred naturally. The percentage of sows standing for the scheduled breeding, farrowing rate and litter size of each group were recorded. Only 60 and 50% of the sows were mated at 0 and 12 h, respectively, after an increase in vaginal mucosal conductivity, while all the sows in the other groups were mated. Sows in the control group and sows bred at 24 and 36 h after the vaginal mucosal conductivity increase had a similar farrowing rate (85%). There was no significant difference for litter size between the treatment groups when compared with those of the controls (0 h:10.33; 12 h:10.8; 24 h:11.11, 36 h:9.76, and controls:10.29;). These results suggest that a single mating at 24 or 36 h after a 15-point or higher vaginal mucosal conductivity increase results in a farrowing rate and litter size which is comparable with twice-mated control sows.

Farrowing induction with cloprostenol-xylazine combination.

Eighty crossbred, multiparous sows, weighing between 190 and 320 kg, were randomly assigned to the following four treatment groups of 20 sows each: 1) saline-saline, 2) cloprostenol-saline, 3) saline-xylazine and 4) cloprostenol-xylazine. The mean gestation length of each multiparous sow was calculated. Cloprostenol (250 ug/sow, i.m.) or saline was given 3 d prior to the calculated due date at 11:30 a.m. Xylazine (2 mg/kg, i.m.) or saline was given 20 h after either the cloprostenol or previous saline treatment. Cloprostenol-xylazine treated sows had the shortest mean farrowing interval (1.5 +/- 0.3 h) when compared with the rest of the treatment groups (saline-saline:66.0 +/- 8.1, cloprostenol-saline:10.5 +/- 1.9, saline-xylazine:60.6 +/- 5.6 h). Farrowing time, percentage of stillbirths, average birth weight, d-5 and d-21 postbirth weights, number of pigs born, number of pigs born alive, and number of pigs surviving at 5 and 21 d afterbirth were not significantly different among the four groups. This study demonstrated that cloprostenol-xylazine treatment decreases the time to onset of farrowing with less variation than cloprostenol or xylazine alone. Therefore, the use of a cloprostenol-xylazine combination is suggested as an alternative method for inducing farrowing.

A Simple Technique for the Purification of Plasma Membranes from Ejaculated Boar Spermatozoa

Spermatozoa were initially separated from fresh boar ejaculates using a 1.0 M sucrose density gradient. Spermatozoa (1 x 10(8) cells/ml) were subjected to gas cavitation (650 psi, 10 minutes), followed by a 4-step centrifugation technique to yield the final plasma membrane preparation. Purity of the plasma membrane isolate was determined using microscopic techniques (i.e. differential interference contrast and transmission electron microscopy) and marker enzymes for biochemical characterization. Plasma membranes were found to be removed primarily from the periacrosomal region of the sperm. Acrosomes appeared to remain intact on the cavitated spermatozoa. Transmission electron microscopy yielded a homogenous population of 100-200 microns unilamellar vesicles. Enzyme markers specific for plasma, acrosome and mitochondrial membranes substantial the purity observed under visual examination.