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Steckler D.,Section of Reproduction | Naidoo V.,University of Pretoria | Gerber D.,Section of Reproduction | Kahn W.,University of Zurich
Theriogenology | Year: 2012

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. © 2012 Elsevier Inc. Source

Steckler D.,Section of Reproduction | Nothling J.O.,Section of Reproduction | Harper C.,Genetics Laboratory
Animal Reproduction Science | Year: 2013

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. © 2013 Elsevier B.V. Source

Steckler D.,Section of Reproduction | Stout T.A.E.,Section of Reproduction | Stout T.A.E.,University Utrecht | Durandt C.,University of Pretoria | Nothling J.O.,Section of Reproduction
Theriogenology | Year: 2015

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 inbicarbonate-free modified Tyrode's medium (-BIC), medium containing 15mM bicarbonate (+BIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6hours. 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 30minutes 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 210minutes 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. © 2015 Elsevier Inc. Source

Heise A.,Section of Reproduction | Thompson P.N.,Section of Reproduction | Gerber D.,Section of Reproduction
Animal Reproduction Science | Year: 2011

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n = 4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-).Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa. © 2010 Elsevier B.V. Source

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