bluuebunny/crossref_metadata_2025 huggingface dataset:
A phase 2a, randomized, double‐blind 28‐day study of TZP‐102 a ghrelin receptor agonist for diabetic gastroparesis
Authors: Ejskjaer N. , Wo J. M. , Esfandyari T. , Mazen Jamal M. , Dimcevski G. , Tarnow L. , Malik R. A. , Hellström P. M. , Mondou E. , Quinn J. , Rousseau F. , McCallum R. W.
Published: December 2012

Background  Gastroparesis causes significant morbidity and treatment options are limited. TZP‐102 a novel, macrocyclic, selective, oral ghrelin receptor agonist, was evaluated in a randomized, double‐blind, placebo‐controlled trial in patients with diabetic gastroparesis.

Methods  A total of 92 outpatients were randomized to once‐daily administrations of 10‐mg (n = 22), 20‐mg (n = 21), 40‐mg (n = 23) TZP‐102 or placebo (n = 26). The primary endpoint was the change from baseline in gastric half‐emptying time (T½) utilizing 13C‐breath test methodology and secondary endpoints included symptom improvement using patient‐reported gastroparesis symptom scores (PAGI‐SYM questionnaire) and patient and physician overall treatment evaluations (OTE).

Key Results  Gastric T½ changes were not statistically significant between TZP‐102 and placebo after 28 days of treatment at any dose. Clinical improvements (−1.0 to −1.4 point mean decrease in symptom severity) occurred in the Gastroparesis Cardinal Symptom Index (GCSI) component of the PAGI‐SYM, which was significant vs placebo for all TZP‐102 doses combined. Improvements became evident after 1 week of treatment. Significantly, more patients given TZP‐102 (any dose) had a 50% reduction in baseline GCSI score (28.8%vs 7.7% placebo). Safety profiles were similar across groups. All TZP‐102 doses were well‐tolerated with no adverse cardiac, weight, or glucose control outcomes.

Conclusions & Inferences  TZP‐102 for 28 days, at doses of 10–40 mg once daily, was well‐tolerated and resulted in a reduction in symptoms of gastroparesis. The lack of correlation between symptom improvement and gastric emptying change is consistent with previous studies in diabetic gastroparesis, and emphasizes the value of patient‐defined outcomes in determining therapeutic benefit.
The Impact of Early PostOperative Pain on Health‐Related Quality of Life
Authors: Taylor Rod S. , Ullrich Kristin , Regan Sophie , Broussard Christina , Schwenkglenks Matthias , Taylor Rebecca J. , Gordon Debra B. , Zaslansky Ruth , Meissner Winfried , Rothaug Judith , Langford Richard ,   The PAIN‐OUT investigators
Published: December 2012

Objectives

To examine how the severity of postoperative pain affects patient's health‐related quality of life (HRQoL) at 1 week following surgery and to compare two generic validated HRQoL instruments.

Methods

Patients undergoing general or orthopaedic surgery at the Royal London Hospital were randomly sampled. The following patient outcome data were collected EQ‐5D (EuroQoL) pre‐operatively and the Revised American Pain Society Patient Outcome Questionnaire (APS‐POQ‐R) at 24 hours postoperation; and EQ‐5D, Short‐Form‐12 (SF‐12) and APS‐POQ‐R at 7 days postoperation. The degree of association between pain and HRQoL was assessed using Pearson's correlation coefficient and multivariate generalized linear regression models.

Results

Of the 228 patients included, 166 patients provided data at 7 days. Sixteen percent reported severe pain ≥ 50% of the day at 7 days. The severity of pain on both the APS‐POQ‐R pain severity and interference and affective impairment domains at 7 days was highly correlated with a decrease in HRQoL as assessed by the SF‐12 Physical Component Score (PCS), SF‐12 Mental Component Score (MCS), and EQ‐5D scores (r = −0.34 to −0.61, P < 0.0001). Multivariate regression analyses showed that irrespective of confounding factors (eg, age, gender, and pre‐operative HRQoL) patients with severe postoperative pain experience important reductions in both physical and mental well‐being domains of their HRQoL.

Conclusions

A proportion of patients continue to experience severe pain at 7 days postoperatively, even after minor surgery. HRQoL is strongly associated with the level of pain and provides additional data on the impact of postsurgery pain on patient's function and well‐being. Additional studies are needed to elucidate the interaction between pain severity and HRQoL during the peri‐operative period.
High‐resolution temperature responses of leaf respiration in snow gum (Eucalyptus pauciflora) reveal high‐temperature limits to respiratory function
Authors: O'SULLIVAN ODHRAN S. , WEERASINGHE K. W. LASANTHA K. , EVANS JOHN R. , EGERTON JOHN J. G. , TJOELKER MARK G. , ATKIN OWEN K.
Published: December 2012

The response of leaf energy metabolism to temperature is central to predicting the impact of environmental gradients and future climate regimes on carbon exchange in forests. Here, we tested whether Eucalyptus pauciflora (an evergreen, broadleaved tree) growing in thermally contrasting environments (including winter‐acclimated trees encased in ice at high altitudes) exhibit generalizable temperature response functions of leaf respiration and fluorescence. We found that leaf energy metabolism was surprisingly heat tolerant (with maximal rates of respiration occurring at 51–57°C), with temperature responses varying seasonally. Collectively, our results: (1) highlight high‐temperature limits of energy metabolism in E. pauciflora; and, (2) provide a framework for improving representation of T‐responses of leaf respiration in predictive models.
In vitro Survival of Follicles Collected from Domestic Cats' Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF‐1
Authors: Alves EA , Padilha L , Savi PA , Apparicio MF , Mostachio GQ , Motheo TF , Pires‐Buttler EA , Vicente WRR , Luvoni GC
Published: December 2012

Contents

Optimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin‐like growth factor (IGF‐1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF‐1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF‐1 (100 ng/ml). Follicles collected during the follicular phase and cultured without IGF‐1 displayed a significant increase in size and higher GC viability (46.5 ± 22.1 μm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF‐1 (26.7 ± 14.4 μm, 50%, respectively; p < 0.05). In contrast, when IGF‐1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF‐1 had a significant increase in their diameter and GC viability (31.9 ± 15.9 μm, 63.6%, respectively) than that cultured without IGF‐1 (26.7 ± 14.4 μm, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF‐1 exerts a positive effect on follicles collected at the luteal phase.
Canine Fibroblasts Expressing Human Transcription Factors: What is in the Route for the Production of Canine Induced Pluripotent Stem Cells
Authors: Gonçalves NJN , Bressan FF , Souza A , Martins DS , Miglino MA , Meirelles FV , Perecin F , Ambrósio CE
Published: December 2012

Contents

The aim of this study was to further clarify the mechanisms involved in inducing pluripotency using canine foetal fibroblast cells. The two pluripotency‐related transcription factors, OCT4 and SOX2, coupled to a fluorescent reporter gene were transduced, individually or in combination, using a lentiviral system. Stable transgenic cell lineages were obtained and canine cells showed to be highly responsive to the integration and expression of human SOX2 and OCT4, also depending on the amount of virus used for incubation. Such positive results are essential for the establishment of pluripotency induction through the incorporation of known transcription factors into the genome of somatic cells.
Chemical Composition of Lipids Present in Cat and Dog Oocyte by Matrix‐Assisted Desorption Ionization Mass Spectrometry (MALDI‐ MS)
Authors: Apparicio M , Ferreira CR , Tata A , Santos VG , Alves AE , Mostachio GQ , Pires‐Butler EA , Motheo TF , Padilha LC , Pilau EJ , Gozzo FC , Eberlin MN , Lo Turco EG , Luvoni GC , Vicente WRR
Published: December 2012

Contents

The aim of the present study was to investigate the level of information on the chemical structures and relative abundances of lipids present in cat and dog oocytes by matrix‐assisted laser desorption mass spectrometry (MALDI‐MS). The MALDI‐MS approach requires a simple analysis workflow (no lipid extraction) and few samples (two or three oocytes per analysis in this work) providing concomitant profiles of both intact phospholipids such as sphingomyelins (SM) and phosphatidylcholines (PC) as well as triacylglycerols (TAG). The lipids were detected in oocytes by MALDI using dihydroxybenzoic acid (DHB) as the matrix. The most abundant lipid present in the MS profiles of bitch and queen oocytes was a PC containing 34 carbons and one unsaturation [PC (34:1)]. Oocytes of these two species are characterized by differences in PC and TAG profiles detected qualitatively as well as by means of principal component analysis (PCA). Cat oocytes were mainly discriminated by more intense C52 and C54 TAG species and a higher number of unsaturations, indicating predominantly linoleic and oleic fatty acyl residues. Comparison of the lipid profile of bitch and queen oocytes with that of bovine oocytes revealed some similarities and also some species specificity: TAG species present in bovine oocytes were also present in bitches and queens; however, a more pronounced contribution of palmitic, stearic and oleic fatty acid residues was noticed in the lipid profile of bovine oocytes. MALDI‐MS provides novel information on chemical lipid composition in canine and feline oocytes, offering a suitable tool to concomitantly monitor, in a nearly direct and simple fashion the composition of phospholipids and TAG. This detailed information is highly needed to the development of improved protocols for in vitro culture and cryopreservation of cat and dog oocytes.
AZA Wildlife Contraception Center Programme for Wild Felids and Canids
Authors: Asa C , Boutelle S , Bauman K
Published: December 2012

Contents

North American zoos began using melengestrol acetate (MGA) implants to control reproduction in wild felids in the mid‐1970s. Research linking MGA and other progestin‐based contraceptives to uterine and mammary pathology in canids as well as felids resulted in a shift to GnRH agonist implants (Suprelorin®: deslorelin, Peptech Animal Health, Australia). However, a recent study revealed an association between Suprelorin® and uterine pathology in canids, but that pathology was not found in canids treated with oral megestrol acetate (MA) for 2 weeks around the time of implant insertion to prevent the initial agonist stimulation phase. Thus, the AZA Wildlife Contraception Center (WCC) currently recommends Suprelorin® plus the 2‐week MA regimen for wild canids and felids. WCC research is now focusing on factors affecting Suprelorin® reversibility.
Leptin and Leptin Receptor Gene Expression in the Canine Corpus Luteum During Diestrus, Pregnancy and after Aglepristone‐Induced Luteolysis
Authors: Balogh O , Kowalewski MP , Reichler IM
Published: December 2012

Contents

Leptin (LEP) and leptin receptor (LEP‐R) expression was shown to change throughout the luteal phase in several species and may be involved in steroid hormone production. In the bitch, leptin but not LEP‐R protein was detected in the non‐pregnant corpus luteum (CL). Until now, no further information has been available on their expression levels and role in CL function. Our objective was to compare time‐related changes in luteal LEP and LEP‐R mRNA levels during the non‐pregnant luteal phase, pregnancy and after aglepristone treatment in mid‐gestation. CLs were collected by ovariohysterectomy at different time points: day (d) 5, 15, 25, 35, 45, 65 after ovulation (p.o.) in non‐pregnant bitches; pre‐implantation, post‐implantation, mid‐gestation, during prepartum luteolysis; 24 and 72 h after aglepristone injection. Non‐pregnant LEP expression was lowest on d5 p.o., increased thereafter and fell again on d45 (P ≤ 0.04). LEP‐R expression was not altered (P = 0.07). In pregnant bitches, neither LEP nor LEP‐R mRNA levels varied over time (P = 0.201 and P = 0.150, respectively). Aglepristone treatment caused substantial downregulation of luteal LEP expression by 72h post‐treatment (P ≤ 0.01). However, LEP‐R expression did not follow the same course (P = 0.193). Our results indicate that both LEP and LEP‐R mRNA are present in the canine CL during the non‐pregnant luteal phase and pregnancy. LEP expression changes significantly over time in non‐pregnant dogs and after aglepristone administration and thus, it may play a role in luteal steroidogenesis and regression.
Prediction of Parturition in Dogs and Cats: Accuracy at Different Gestational Ages
Authors: Beccaglia M , Luvoni GC
Published: December 2012

Contents

In bitches and queens, the ultrasonographic measurement of extrafoetal and foetal structures allows the evaluation of gestational age and the prediction of the parturition term for an extended period of time. The aim of this study was to investigate whether the accuracy of parturition date prediction is affected by the week of pregnancy when the ultrasonographic examination is performed. The results were obtained by retrospective analysis on the gestational period basis (from week 4 to week 9 of pregnancy) in 495 ultrasonographic examinations of pregnant bitches (small and medium size) and 60 of pregnant queens. They demonstrated that a similar accuracy (p > 0.05) was obtained by the measurement of inner chorionic cavity at week 4 and 5 of pregnancy (±1 day, 81% vs 67.7%; ±2 days, 93.1% vs 85.9%). Accuracy (±1 day) based on biparietal (BP) measurement was similar at week 5 and 6 of pregnancy (78.6% vs 78.9%; p > 0.05), whereas a significant decrease (p < 0.05) was observed at week 7 and 8 compared to week 6. No difference in the accuracy of the prediction at ±2 days based on BP was observed from week 5 to 8 of gestation (range from 82.3% to 95.2%). Close to term (week 9), the accuracies of the prediction based on BP were significantly lower than those obtained at week 5 (±1 day: 50.9% vs 78.6%, p < 0.05; ±2 days: 69.8% vs 95.2%, p < 0.005). The accuracy of the prediction in bitches and queens is influenced by the gestational period when ultrasonographic measurements are performed, particularly for BP measurement. Although a decrease in BP accuracy occurs along the pregnancy, the accuracy at ±1 and ±2 days of prediction of parturition term is highly consistent for 6 and 8 weeks of gestation, respectively.
Are Oocytes from the Anestrous Bitch Competent for Meiosis?
Authors: Chastant‐Maillard S , Saint‐Dizier M , Grimard B , Chebrout M , Thoumire S , Reynaud K
Published: December 2012

Contents

In the bitch, oocyte meiosis resumption takes place in the oviduct. Using oocytes from anestrous bitches, in vitro maturation (IVM) generally gives very poor results. To investigate the contribution of oocyte competence to the low IVM yield, we compared in vivo maturation in an optimal environment with conventional IVM. A total of 418 grade 1 cumulus‐oocyte complexes (COCs) from 10 anestrous bitches were transferred into the oviducts of recipient bitches either on Day −1 (n = 3 recipients), Day 0 (n = 2) or on Day +1 (n = 2) relative to ovulation. For each donor bitch, 20 grade 1 COCs were also cultured in vitro. After 72 h of in vivo or IVM, the nuclear stage of oocytes was determined after DNA and tubulin staining. Of the 154 oocytes recovered and examined after intratubal transfer, only 2% reached the metaphase I or II stage and 38.3% were degenerated. Oocytes cultured in vitro displayed a higher metaphase rate (7.6%, n = 170) and lower degeneration rate (12.9%) compared with transferred oocytes (p < 0.001). These results clearly demonstrate that the oocyte competence is the major limiting factor of IVM efficiency in the dog. Mimicking the tubal environment may thus not be sufficient to increase IVM yield in this species.
Timing of the Intestinal Barrier Closure in Puppies
Authors: Chastant‐Maillard S , Freyburger L , Marcheteau E , Thoumire S , Ravier JF , Reynaud K
Published: December 2012

Contents

As puppies are born with very low immunoglobulin concentrations, they rely on passive immune transfer from ingested colostrum to acquire a protective immunity during the first few weeks of life. The purpose of this study was to describe the timing of gut closure in canine neonates. Twenty‐twoBeagle puppies received 3 ml of standardized canine colostrum at 0, 4, 8, 12, 16 or 24 h after birth using a feeding tube. Blood immunoglobulinsG(IgG,MandA) were assayed 0, 4 and 48 h after colostrum ingestion.IgGabsorption rate was significantly affected by the time of colostrum administration, and theIgGconcentrations in puppies serum 48 h after administration were significantly higher when colostrum was ingested at 0–4 h of age than at 8–12 h or 16–24 h (1.68 ± 0.4, 0.79 ± 0.07 and 0.35 ± 0.08 g/l, respectively; p < 0.001). In the canine species, gut closure seems thus to begin as early as 4–8 h after birth and to be complete at 16–24 h. Consequently, this phenomenon appears to occur earlier in puppies than in most other species.
Effects of Cold Storage Prior to Freezing on Superoxide Dismutase, Glutathione Peroxidase Activities, Level of Total Reactive Oxygen Species and Sperm Quality in Dogs
Authors: Chatdarong K , Chaivechakarn A , Thuwanut P , Ponglowhapan S
Published: December 2012

Contents

Reactive oxygen species (ROS) are one of several crucial factors that cause mammalian sperm damage during handling and preservation. Their deleterious effects can be restricted by the action of antioxidants. The present study aimed at investigating: (i) effects of cold storage prior to freezing on activities of enzymatic antioxidants (superoxide dismutase; SOD and glutathione peroxidase; GPx) and level of total reactive oxygen species (tROS); and (ii) effects of SOD or SOD plus GPx supplementation to chilled (3 and 96 h) and frozen‐thawed semen. Six privately owned dogs were included. Experiment I: Each pooled semen was divided into three equal aliquots, which were subjected to chilled storage for 3, 24 or 96 h prior to freezing (n = 7). The activities of SOD and GPx in sperm cells and tROS level in chilled and frozen‐thawed semen were measured. Experiment II: Pooled semen was divided to be cold stored for 3 or 96 h in three different extenders; (i) Uppsala Equex extender (control), (ii) Uppsala Equex extender supplemented SOD, or (iii) Uppsala Equex extender supplemented with SOD plus GPx. Sperm motility, viability and integrity of acrosome and DNA was evaluated after cold storage and frozen‐thawed. The cold storage from 24 h prior to freezing resulted in a decrease in the SOD activity in the frozen‐thawed sperm cells whereas the GPx activity and tROS levels were not affected. In addition, the supplementation of SOD plus GPx enhanced the percentage of sperm viability and DNA integrity after cold stored and frozen‐thawed. In sum, the SOD activity is compromised by cold storage prior to freezing of dog semen. Addition of GPx is suggested to assist SOD to complete the enzymatic ROS scavenging system in the dog sperm.
On the Horizon for Fertility Preservation in Domestic and Wild Carnivores
Authors: Comizzoli P , Wildt DE
Published: December 2012

Contents

Innovations are emerging from the growing field of fertility preservation for humans and laboratory animals that are relevant to protecting and propagating valuable domestic and wild carnivores. These extend beyond the ‘classical’ approaches associated with sperm, oocyte and embryo freezing to include gonadal tissue preservation combined with in vitro culture or xenografting, all of which have potential for rescuing vast amounts of unused and wasted germplasm. Here, we review approaches under development and predicted to have applied value within the next decade, including the following: (i) direct use of early‐stage gametes for in vitro fertilization; (ii) generation of more mature gametes from gonadal tissue or stem cells; (iii) simplification, enhanced safety and efficacy of cryopreservation methods; and (iv) biostabilization of living cells and tissues at ambient temperatures. We believe that all of these fertility preservation strategies will offer knowledge and tools to better manage carnivores that serve as human companions, valuable biomedical models or require assistance to reverse endangerment.
Ex vivo Recovery of Preimplantatory Embryos in Bitches
Authors: Commin L , Buff S , Rosset E , Joly T , Guerin P , Neto V
Published: December 2012

Contents

The present study was conducted to investigate the timing of preimplantatory development in the dog and to evaluate the efficiency of flushing oviducts and uterine horns to collect embryos. Among the embryonic structures collected between day 8 and day 12 after ovulation, 43 % were at the 1–16 cells stage, 23% were at the morula stage and 34% at the blastocyst stage. Our collection method yielded to a recovery rate of 61.3 %, and 7.1 ± 0.7 embryos were harvested per bitch. In addition, the ovulation rate reached 11.6 ± 0.8 per bitch. The first morulae were observed from day 9 post‐ovulation, while the first blastocyst appeared from day 10. Two‐thirds of the collected morulae‐blastocysts were obtained between the 11th and the 12th day after ovulation. To the moment, we suggest this is the best period to harvest canine embryo for cryopreservation.
Factors Influencing Annual Fecal Testosterone Metabolite Profiles in Captive Male Polar Bears (Ursus maritimus)
Authors: Curry E , Roth TL , MacKinnon KM , Stoops MA
Published: December 2012

Contents

The objectives of this study were to assess the effects of season, breeding activity, age and latitude on fecal testosterone metabolite concentrations in captive, adult male polar bears (Ursus maritimus). Fourteen polar bears from 13 North American zoos were monitored for 12–36 months, producing 25‐year‐long testosterone profiles. Results indicated that testosterone was significantly higher during the breeding season (early January through the end of May) compared with the non‐breeding season with the highest concentrations excreted from early January through late March. Variations in excretion patterns were observed among individuals and also between years within an individual, with testosterone peaks closely associated with breeding activity. Results indicate that fecal testosterone concentrations are influenced by season, breeding activity and age, but not by latitude. This is the first report describing longitudinal fecal testosterone metabolite concentrations in individual adult male polar bears.
Sperm Nuclear Decondensation Induction Capacity of In Vitro and In Vivo Matured Canine Oocytes
Authors: De los Reyes M , Palomino J , Parraguez VH , Vergara J
Published: December 2012

Contents

The objectives of this study were to evaluate the sperm nuclear decondensation capacity of ovulated and in vitro‐matured (IVM) canine oocytes during different culture times and correlate this decondensation ability with the state of oocyte nuclear maturation in vitro and in vivo. Fresh ejaculates from three dogs were used for in vitro fertilization (IVF). Dog spermatozoa were cocultured with ovulated or IVM oocytes after each culture period (0, 48, 72 and 96 h) for 24 h. The nuclear stage of the oocytes and the appearance of the sperm nucleus were determined, and data were analysed with a chi‐square test. The rates of decondensation and meiotic development in IVM oocytes increased up to 72 h of culture. In contrast, almost all in vivo‐matured oocytes showed MII nuclear stage and sperm chromatin decondensation. The percentages of oocytes at MII stage were much lower (p < 0.05) in all IVM groups compared with ovulated oocytes; the rate of sperm chromatin decondensation was higher in ovulated oocytes than in those matured in vitro. Thus, IVM canine oocytes are able to decondense the sperm chromatin during IVF, and this ability increases with time. Nevertheless, sperm chromatin decondensation is less efficient than in ovulated oocytes and may not be completely synchronized with nuclear development as it occurs in vivo.
Golgi Apparatus and Endoplasmic Reticulum Dynamic during Meiotic Development in Canine Oocytes
Authors: De los Reyes M , Palomino J , Jofré S , Villarroel A , Moreno R
Published: December 2012

Content

The Golgi apparatus (GA) and endoplasmic reticulum (ER) play a central role in the events related to intracellular trafficking distribution. This work evaluated the dynamics and localization of the GA and ER in canine oocytes during meiotic development in vitro. Cumulus–oocytes complexes (COCs) from ovaries of adult bitches were incubated for IVM for 0, 48, 72 and 96 h. At each time, the nuclear status was determined using DAPI staining, and the GA was evaluated by immunofluorescence using two antibodies against Golgi proteins: GM130 and Giantin. ER was analysed with fluorescent lipid probes (ER‐Tracker), for living cells. Golgi structures were homogeneous in the cytoplasm in non‐matured oocytes, mainly in those GV‐arrested oocytes. In contrast, at 48 h and from GVBD stage, the immunolocalization began to be subcortical, increasing at 72 h and 96 h. Meiotic development increased with time and the majority of oocytes at MI‐MII stages showed cortical distribution of Golgi structure. Living ZP intact non‐matured oocytes showed a reticular pattern of ER that covered oocyte cortex. Confocal microscopy showed that, in all levels cuts the fluorescence marks were located in the cortical region, irrespective of culture time. The changes and localization in these organelles during IVM might be related to meiotic development, but in a non‐synchronous manner.
Progesterone (PR), Oestrogen (ER‐α and ER‐β) and Oxytocin (OTR) Gene Expression in the Oviduct and Uterus of Pregnant and Non‐pregnant Bitches
Authors: Derussi AAP , de Souza RWA , Volpato R , Guaitolini CRF , Ackermann CL , Taffarel MO , Cardoso GS , Dal‐Pai‐Silva M , Lopes MD
Published: December 2012

Contents

The aim was to assess hormone receptor gene expression in the oviduct and uterus during canine pregnancy. Nineteen pregnant bitches divided into four groups were ovariohysterectomized (OVH) at either day 8, 12, 21 or 60 of pregnancy, and five non‐pregnant females underwent OVH 12 days after the pre‐ovulatory Luteinizant Hormone (LH) surge and served as controls. RT‐qPCR for progesterone (PR), oestrogen (ER‐α and ER‐β) and oxytocin (OTR) receptors was performed on the oviduct and uterine tissue. The mRNA PR expression in the uterus during early stages of pregnancy and the luteal phase was higher than at other times. The mRNA ER‐β expression in the oviduct during early pregnancy was less than in non‐pregnant bitches. In the uterus, the mRNA ER‐β expression was higher in the initial stages of pregnancy. The ER‐α expression was higher in the oviduct and uterus in advanced stages of pregnancy. The mRNA OTR expression in the oviduct was lower than in the uterus in control group. The expression of this receptor in oviduct and the uterus was higher in the final stages of pregnancy, when compared with other phases. These data suggested that the serum progesterone concentrations probably exert a direct control on the PR and ER (α and β) expression and indirectly on OTR expression in the bitch oviduct and uterus.
Applying Gene Silencing Technology to Contraception
Authors: Dissen GA , Lomniczi A , Boudreau RL , Chen YH , Davidson BL , Ojeda SR
Published: December 2012

Contents

Population control of feral animals is often difficult, as it can be dangerous for the animals, labour intensive and expensive. Therefore, a useful tool for control of animal populations would be a non‐surgical method to induce sterility. Our laboratories utilize methods aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A useful framework for design of a new approach will be the combination of these methods with the intended goal to produce a technique that can be used to non‐invasively sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: the target gene must be essential for fertility; the method must include a mechanism to effectively and specifically silence the gene of interest; the method of delivering the silencing agent must be minimally invasive, and finally, the silencing effect must be sustained for the lifespan of the target species, so that expansion of the population can be effectively prevented. In this article, we discuss our work to develop gene silencing technology to induce sterility; we will use examples of our previous studies demonstrating that this approach is viable. These studies include (i) the use of viral vectors able to disrupt reproductive cyclicity when delivered to the regions of the brain involved in the control of reproduction and (ii) experiments with viral vectors that are able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy.
Physiologic Responses Following Gonadotropin‐Releasing Hormone Immunization in Intact Male Dogs
Authors: Donovan CE , Greer M , Kutzler MA
Published: December 2012

Contents

We investigated the use of a commercial gonadotropin‐releasing hormone (GnRH) vaccine as a method of temporary and reversible immunocastration in intact male dogs. Four privately owned dogs were vaccinated twice at 4‐week intervals. Blood samples were collected at 0, 4, 12 and 20 weeks following the initial vaccination. These samples were analysed forGnRHantibody titres, luteinizing hormone (LH) and testosterone concentrations. Scrotal measurements were made at the time of sample collection, and testicular volume was calculated using the formula of an ellipsoid. As a result of vaccination, dogs displayed an elevatedGnRHantibody titre, decreasedLHand testosterone concentrations and decreased testicular volume, which reversed by the end of the study period. Therefore, these results suggest that immunizing againstGnRHmay be a possible choice for temporary and reversible immunocastration.
Effects of Cooling Time on Membrane Integrity and Motility of Frozen‐Thawed Canine Spermatozoa using Two Different Commercial Egg Yolk–based Extenders at Two Different Cooldown Equilibration Times
Authors: du Bois S , Len JA , Parlevliet JM , Eilts BE
Published: December 2012

Contents

Two commercially available egg yolk–based semen extenders, one marketed for human semen freezing (HEYE) and one marketed for canine semen freezing (CEYE), were used to cryopreserve semen from single ejaculates of 11 different dogs. For each extender, a 30‐ and a 60–min cooldown period was used prior to the addition of the extender containing glycerol and then immediately frozen in liquid nitrogen vapours. Sperm motility was measured using a computer‐assisted semen analysis (CASA) system. Sperm intact membranes were measured using SYBER‐14 and propidium iodide. Semen in the HEYE cooled for 60 min had a significantly greater percentage of intact membranes than the semen in the HEYE cooled for 30 min (p = 0.02). Semen in the HEYE cooled for 60 min had significantly greater total motility (p = 0.007) and progressive motility (p = 0.004) than semen cooled for 60 min in the CEYE and semen cooled for 30 min in the HEYE (total motility p = 0.02 and progressive motility p = 0.02). Semen cooled for 60 min in the CEYE did not differ significantly in total (p = 0.6) or progressive motility (p = 0.4) than semen cooled for 30 min in the CEYE. There was no difference in total (p = 0.8) or progressive motility (p = 0.8) between the semen cooled for 30 min in the HEYE and the semen cooled for 30 min in the CEYE.
Stimulation of Mating‐Induced Uterine Contractions in the Bitch and Their Modification and Enhancement of Fertility by Prostatic Fluid
Authors: England GCW , Moxon R , Freeman SL
Published: December 2012

Contents

Little is known about the response of the bitch's reproductive tract to mating or of the role of male accessory gland secretions in the female. In this clinical study, the component stimuli causing mating‐induced uterine contractions were investigated in 64 bitches. Basal uterine contractions were present during oestrus and a significant increase in the frequency of contractions was observed during natural mating. Neither teasing with a male nor stimulation of the vagina or cervix by vaginal or transcervical insemination (TCI) caused an increase in the frequency of uterine contractions. Increased contractions were however present after both vaginal and transcervical insemination when the vestibule was distended, and dorsal wall of the vaginal was manually stimulated. Interestingly, this increase in uterine contractions was partially ameliorated when prostatic fluid was used as a flushing component following transcervical insemination. Two further studies performed with 72 bitches of which 18 were each inseminated transcervically with fresh or frozen semen flushed into the uterus with either saline or prostatic fluid demonstrated that prostatic fluid significantly increased the pregnancy rate and litter size of both groups. There are important mechanisms regulating the transport and elimination of sperm from the bitch's reproductive tract. Whilst physical aspects of coitus are undoubtedly involved in initiating uterine contractions, prostatic fluid appears to have an important role in modulating uterine contractions and fertility.
Customizing Semen Preservation Protocols for Individual Dogs and Individual Species: Sperm Preservation beyond the State of the Art
Authors: Farstad W
Published: December 2012

Contents

Sperm quality can be variable in morphometric and physiological attributes between males of different species, between males within species subtypes reared under different environmental conditions, between ejaculates of the same male or even between sperm populations within an ejaculate. Clinical semen evaluation is based on evaluation of whole ejaculates, which is not a chemically or physiologically well‐defined entity, rather a collection of heterogeneous subpopulations giving different measurements and possessing different fertilizing potential. Identification of subpopulations with different motility patterns is important as well as characterizing the subtle structural changes underlying the motility differences observed. The ability to identify populations of sperm responding rapidly or failing to progress through the capacitation process may have clinical applications. Studies of lipid‐phase fluidity of sperm membranes, mathematical modelling of membrane ion transport, role of modifying components and detergent‐resistant microdomains are of particular interest. When customizing extenders to ejaculates from cryosensitive males or species, a thorough knowledge of species sperm membrane physiology and an assessment of the individual ejaculate's sperm populations are necessary. Structural differences have been found in sperm membranes between fox species with different cryosurvival potential of their spermatozoa. Supplementation of lipids and detergents in cryoextenders may influence membrane fluidity of the surviving spermatozoa in a species‐dependent manner and influence capacitation. Immobilization of sperm prior to cryopreservation with subsequent slow release of sperm in the female genital tract may be a way to prolong the fertile life of sperm. In canids with a long oocyte maturation time, delayed capacitation may be beneficial.
Gene Expression in Pre‐Term, Pre‐Labour and Parturient Canine Placenta
Authors: Fellows EJ , Hazzard TM , Kutzler MA
Published: December 2012

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Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), vascular endothelial growth factor (VEGF)‐A, VEGF‐A receptor (Flt‐1) and KiSS‐1 receptor (KiSS‐1R) all play a role in trophoblast invasion in a number of mammalian species. However, mRNA expression of factors regulating trophoblast invasion has not been studied in dogs. Abnormal expression of these factors at the end of canine gestation may contribute to placental retention and/or subinvolution of placental sites. Therefore, we sought to determine the relative mRNA expression of these factors in canine chorioallantois tissue at 61 ± 1 day past the LH surge (pre‐term; n = 4), following elective c‐section at 64 ± 1 day past the LH surge prior to first‐stage labour (pre‐labour; n = 4) and following natural delivery (parturient; n = 3). Total RNA was isolated, real‐time RT‐PCR was performed, and relative expression was calculated using the relative quantitation () method. MMP‐9 mRNA expression was significantly higher in pre‐term samples compared to pre‐labour and parturient samples. The results showed no significant difference between MMP‐2, TIMP‐2, VEGF‐A and Flt‐1 mRNA expression among the three groups. KiSS‐1R mRNA was not expressed in any tissues studied. Gene expression of MMP‐9 may be related to the onset of labour, whereas MMP‐2, VEGF‐A, Flt‐1, TIMP‐2 and KiSS‐1R mRNA do not appear to play a role at the end of gestation in the dog.
Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein‐Free Medium
Authors: Fujihara M , Comizzoli P , Wildt DE , Songsasen N
Published: December 2012

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Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α‐minimum essential medium (α‐MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock‐out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein‐free α‐MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24‐well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α‐MEM compared to 37.5% of those incubated for 15 days in MEM in protein‐free condition (p < 0.05). In contrast, α‐MEM was superior (p < 0.05) to MEM in maintaining dog follicle viability (32.7% vs 8.1%) in protein‐free condition at 15 days. Serum was detrimental (p < 0.05) to follicle survival in both species. Knock‐out serum replacement supplementation and a protein‐free condition supported cat follicle viability, whereas the latter was superior (p < 0.05) to the former for sustaining dog follicle survival. Likewise, dog follicle viability was enhanced (p < 0.05) by the agarose gel compared to the cell culture insert and control groups after 3 and 9 days of culture. For the cat, the agarose gel better (p < 0.05) supported follicle viability compared to the control, but was equivalent to the cell culture insert. Therefore, sustaining primordial follicle survival from intracortical ovarian slices requires a different in vitro microenvironment for the cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange.
Efficacy of Cloprostenol or Aglepristone at 21–22 and 35–38 Days of Gestation for Pregnancy Termination in Queens
Authors: García Mitacek MC , Stornelli MC , Praderio R , Stornelli MA , de la Sota RL
Published: December 2012

Contents

The objective of this study was to evaluate the efficacy of cloprostenol (CLO) or aglepristone (ALI) for pregnancy termination in queens at 21–22 and 35–38 days of gestation. Two experiments (EXP) were carried out to accomplish this aim. Thirty‐seven 12‐ to 14‐month‐old mixed breed queens were used in a randomized design. At oestrus, queens were housed for mating with a tom, and pregnancy was confirmed by transabdominal ultrasonographic examination (US). On days 21–22 of pregnancy (EXP1) or 35–38 of pregnancy (EXP2), queens were divided into three groups (G). Queens in G1 received ALI (10 mg/kg, sc; EXP1, n = 6; EXP2, n = 6) on two consecutive days. Queens in G2 received CLO (5 μg/kg, sc; EXP1, n = 6; EXP2 = 7) on three consecutive days. Queens in G3 received 1 ml of saline solution (PLA, sc; EXP1, n = 6; EXP2 = 6). After treatment, females were monitored daily by US during for 10 days and weekly until the end of gestation. In EXP1, pregnancy was terminated in (6/6, 100%), (0/6, 0%) and (0/6, 0%), for the ALI, CLO and PLA groups, respectively (p < 0.001). In EXP2, pregnancy was terminated in (6/6, 100%), (1/7, 14%) and (0/6, 0%) for the ALI, CLO and PLA groups, respectively (p < 0.001). In both EXP, after CLO administration, animals vomited and were depressed for 30 min; but no side effects were observed in the animals in the ALI group. In conclusion, the results from this study indicate that three injections of CLO are not effective, but two injections of ALI are effective to induce abortion in queens at 21–22 or 35–38 days of pregnancy.
Unilateral Orchidectomy in Mature Cats is Not Followed by Compensatory Hypertrophy
Authors: Romero GG , Barbeito C , Fernandez P , Gimeno E , Gobello C
Published: December 2012

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The aim of this study was to describe the effect of unilateral orchidectomy on testicular characteristics of mature domestic cats. Five, 1‐ to 2‐year‐old, cross‐bred male cats were unilaterally orchidectomized on day 0 (right) and day 60 (left). The testes were processed for histological and immunohistochemical evaluation and groups compared by Student's t‐test. No significant differences (p > 0.05) were found between testes groups for any of the gross (volume and gonadosomatic index) and microscopic (tubular diameters, axis, area, length and perimeter, germinal epithelium height, spermatogonias, spermatocytes, spermatids, spermatozoa, Sertoli and Leydig cells, intertubular compartment, lumen, cellular debris, tubular‐ intertubular proportion and basement membrane) parameters assessed. According to these biometric and morphometric results, mature cats do not develop compensatory hypertrophy within 60 days of unilateral orchidectomy.
Effects of GnRH Antagonists vs Agonists in Domestic Carnivores, a Review
Authors: Gobello C
Published: December 2012

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Gonadotrophin‐releasing hormone (GnRH) stimulates the pituitary secretion of both luteinizing and follicle‐stimulating hormones, and thus controls the hormonal and reproductive functions of the gonads. GnRH analogs, which include agonists and antagonists, have been produced by amino acid substitutions within the native GnRH molecule resulting in greater potency and a longer duration of effectiveness. While the initial antagonists produced significant side effects, more recent potent, long‐acting, water‐soluble, low histamine‐release third‐generation compounds such as cetrorelix, abarelix, azaline B and acyline have appeared. Differently to GnRH agonists, antagonists competitively block and inhibit GnRH‐induced GnRH receptor gene expression leading to an immediate, dose‐dependent, pituitary suppression without an initial stimulation of the gonadal axis. The aims of this review are to compare the effects of GnRH agonists vs antagonists and to describe the existing literature concerning new antagonists in domestic carnivores. In male dogs, a single subcutaneous dose of acyline safely and reversibly decreased serum gonadotrophins and testosterone concentrations for 9 days and prevented physiological response of gonadal the axis to agonistic challenge for 14 days. The same protocol reversibly impaired spermiogenesis, spermatocytogenesis and semen quality in both cats and dogs. In females, third‐generation GnRH antagonists prevented ovulation and interrupted pregnancy in canids but not in felids. During anestrus, a single acyline injection exhibited limited prevention of the ‘flare‐up’ effect in GnRH agonist‐implanted bitches. Although GnRH antagonists appear to have a promising future in domestic carnivores reproduction, the information is still scarce and further work is needed before they can be widely recommended.
Embryo Retrieval after Hormonal Treatment to Control Ovarian Function and Non‐surgical Artificial Insemination in African Lions (Panthera leo)
Authors: Goeritz F , Painer J , Jewgenow K , Hermes R , Rasmussen K , Dehnhard M , Hildebrandt TB
Published: December 2012

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Assisted reproduction technologies are essential for propagating endangered wild felids. Artificial insemination (AI) has been reported in several wild feline species, but pregnancy rates are low, partially owing to failures of current hormonal stimulation protocols. Therefore, this study describes the application of reliable methods to monitor ovarian activity and the development of an effective hormonal protocol to induce oestrus and ovulation in African lions. Application of porcine FSH and porcine LH was shown to be effective for inducing follicular growth and ovulation, and this regimen appeared to be superior to protocols described earlier in terms of ovulation and fertilization rates. Furthermore, non‐surgical AI was performed successfully in lions, and uterine‐stage embryos were collected and cryopreserved. African lions may serve as a valuable model to develop assisted reproduction for propagation of relic zoo populations in the critically endangered Asian lion or Barbary lion.
Scriptaid and 5‐aza‐2′deoxycytidine Enhanced Expression of Pluripotent Genes and in vitro Developmental Competence in Interspecies Black‐Footed Cat Cloned Embryos
Authors: Gómez MC , Biancardi MN , Jenkins JA , Dumas C , Galiguis J , Wang G , Earle Pope C
Published: December 2012

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Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black‐footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but ‘inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent‐related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black‐footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black‐footed cat donor cells and reconstructed embryos with epigenetic‐modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.