Use of contrast-enhanced ultrasonography in chronic pathologic canine testes.

Contrast-enhanced ultrasound with sulphur hexafluoride microbubbles was performed in seven healthy dogs without a history of reproductive pathology and with histologically confirmed normal testes and in 42 dogs with chronic scrotal anomalies. All dogs underwent orchiectomy and histological examination. Enhancement patterns and perfusion parameters (peak intensity and regional blood flow) of testes of healthy dogs and testes with chronic lesions were compared. Fourteen non-pathologic and 60 pathologic testes were considered. Forty testes were neoplastic (24 interstitial cell tumours, 9 seminomas, 7 Sertoli cell tumours), 20 were non-neoplastic (16 testicular degenerations, 2 chronic orchitis, 1 testicular atrophy, 1 interstitial cell hyperplasia). In healthy dogs, the contrast medium flow had a rapid homogeneous wash-in and wash-out, with a short peak phase. With contrast ultrasound, testes that were inhomogeneous with a hyperenhancing pattern were associated with neoplasia (sensitivity: 87.5%, specificity: 100%). Lesions with persistent inner vessels and a hypo-to-isoechoic background were significantly associated with seminomas (sensitivity: 77.8%, specificity: 100%). Testes with non-neoplastic lesions were characterized by a scant/moderate homogeneous enhancement. Perfusion parameters were higher in neoplastic lesions. Contrast ultrasound was a feasible diagnostic tool in the assessment of testicular lesions, with hyperenhancement being an important feature in the diagnosis of malignancy.

Stimulation of mating-induced uterine contractions in the bitch and their modification and enhancement of fertility by prostatic fluid.

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.

B-mode and contrast-enhanced ultrasonographic findings in canine prostatic disorders.

Ultrasound is a common imaging technique used for examination of the dog's prostate gland that has proven to be successful for diagnosis of many clinical conditions. Whilst B-mode ultrasound is useful for assessment of the prostate gland, there appears to be substantial variation in the appearance of some pathological conditions, although individual experienced ultrasonographers often have a high aptitude for making a correct diagnosis. Here, we report the normal volume and appearance of the canine prostate gland with B-mode ultrasound and use thematic analysis to identify the categories described by experienced ultrasonographers when reporting both normal and abnormal findings. Four thematic categories were identified: background echotexture, parenchymal stippling, generalized appearance and focal changes. We found that a relatively narrow variety of thematic variables (descriptors) were used for reporting of prostatic pathology within these thematic categories. There was also poor association between these descriptors and the underlying pathology, with the only unique descriptors being 'cysts' for benign prostatic hyperplasia and 'parenchymal mineralization' for adenocarcinoma. In comparison with the limitations of B-mode ultrasound, we document the value of measurement of vascular perfusion kinetics using contrast-enhanced ultrasound and demonstrate how perfusion kinetics differ between benign and malignant prostatic disease.

Normal and abnormal response to sperm deposition in female dogs: A review and new hypotheses for endometritis.

In mammalian species there are significant physiological responses of the female reproductive tract to the deposition of sperm. These are particularly notable in species where sperm are deposited directly into the uterus, and function both to facilitate sperm transport to the sperm reservoir, and to eliminate introduced contaminants. In the bitch, sperm are deposited into the vagina and are rapidly transported through the open cervix. Sperm are then distributed around the uterus by uterine contractions such that transportation to the tip of the uterine horns occurs within 1 min of the start of mating. The main sperm reservoir appears to be the distal part of the utero-tubal junction which forms a pre-uterine tube reservoir. Sperm remain attached here by their heads to uterine epithelium and remain viable. In non-capacitating conditions sperm slowly detach from this site and this seems important to replenish the uterine tube reservoir, where sperm may re-attach to the epithelium. Post-ovulatory signals trigger capacitation changes and subsequent hyperactivated motility that is associated with detachment of sperm from both reservoirs; thus facilitating fertilization. After mating, a physiological post-mating uterine inflammatory response occurs, evidenced by an influx of polymorphonuclear neutrophils, increased uterine contractions, an increased uterine artery blood flow and a decrease of the resistance index indicating a short-duration vasodilation. Disturbance of this tightly regulated system has the potential to impact fertility by a failure of elimination of the introduced contaminants (such that a clinically-significant post-breeding endometritis ensues) but also by impairing sperm transport.

Horse owners' knowledge, and opinions on recognising colic in the horse.

BACKGROUND: Colic is the most common emergency problem in the horse. An owner's ability to recognise colic and seek assistance is a critical first step in determining case outcome. OBJECTIVES: The aim of this study was to assess horse owners' knowledge and opinions on recognising colic. STUDY DESIGN: Cross-sectional study. METHODS: An online questionnaire was distributed to horse owners with open and closed questions on their knowledge of normal clinical parameters in the horse, confidence and approach to recognising colic (including assessment through case scenarios), and their demographics. Descriptive and chi squared statistical analyses were performed. RESULTS: There were 1564 participants. Many respondents either did not know or provided incorrect estimates for their horse's normal clinical parameters: only 45% (n = 693/1540) gave correct normal values for heart rate, 45% (n = 694/1541) for respiratory rate and 67% (n = 1028/1534) for rectal temperature. Knowledge of normal values was statistically associated with participants' educational qualifications (P<0.01). Owners said if they suspected their horse had colic they would assess faecal output (76%; n = 1131/1486), gastrointestinal sounds (75%; n = 1113/1486), respiratory rate (65%; n = 967/1486) and heart rate (54%; n = 797/1486). There was a lack of consensus on whether to call a vet for behavioural signs of colic, unless the signs were severe or persistent. The majority of participants (61%) were confident that they could recognise most types of colic. In the case scenarios, 49% were confident deciding that a surgical case had colic, but 9% were confident deciding an impaction case had colic. MAIN LIMITATIONS: Most respondents were UK based; risk of self-selection bias for owners with previous experience of colic. CONCLUSIONS: There was marked variation in horse owners' recognition and responses to colic, and significant gaps in knowledge. This highlights the need for the development of accessible educational resources to support owners' decision-making.

Follicular dynamics, ovulation and conception rates in bitches.

Real-time ultrasound imaging was used in a clinical study to estimate the number of follicles of different sizes, ovulation and conception rates, and to study follicle dynamics following oestrus-induction of bitches. Follicles were identified during late anoestrus (between 100 and 60 days prior to the pre-ovulatory LH surge) and there appeared to be a shift in the population from small follicles (1-3 mm in diameter) to large follicles (>4 mm diameter) approximately 2 days prior to the LH surge. Corpora lutea could be reliably identified although the majority were cavitated. High ovulation rates (97-100%) and pregnant rates (86-100%) were detected, and although the conception rate was approximately 70% it varied between 8 and 92%. Within the narrow range of the clinical population studied there were trends relating age to reproduction performance. Oestrus induction with a gonadotrophin regime appeared to result in large numbers of small follicles that did not ovulate, whilst when using cabergoline the number of small and large follicels and the number of copora lutea were similar to those of control cycles.

Prostatic perfusion in the dog using contrast-enhanced Doppler ultrasound.

Ultrasonography has become the imaging modality of choice for evaluation of the prostate gland in the dog. Unfortunately, despite providing excellent images, it may be difficult to differentiate the common canine prostatic diseases with ultrasound because many have a similar ultrasonographic appearance. Real-time contrast-enhanced ultrasound was used to monitor and characterise the normal perfusion pattern and perfusion dynamics of the canine prostate gland when using a micro bubble contrast agent. In all contrast studies, the prostatic artery, entered the prostate gland on the dorso-lateral surface then tunnelled into the prostatic capsule and branched into many small parenchymal arteries which were directed medially towards the urethra to supply the body of the prostate gland. The flow of the contrast medium into the prostatic parenchyma was visible after 15 s. During the wash-in phase, there was an homogenous enhancement of the prostatic parenchyma. During the wash-out phase, an homogenous decrease of the echogenicity was visible in all cases.

Development and validation of a new standardised data collection tool to aid in the diagnosis of canine skin allergies.

Canine atopic dermatitis (cAD) is a common hereditary clinical syndrome in domestic dogs with no definitive diagnostic tests, which causes marked morbidity and has a high economic impact internationally. We created a novel questionnaire for Labrador (LR) and Golden retriever (GR) owners to evaluate canine skin health with respect to clinical signs of cAD. 4,111 dogs had fully completed questionnaires (2,803 LR; 1,308 GR). 'Cases' (793) had a reported veterinary diagnosis of cAD, and 'controls' (1652) had no current or past clinical signs of cAD and were aged >3 years. Remaining dogs (1666) were initially categorised as 'Other'. Simulated annealing was used comparing 'Cases' and 'Others' to select a novel set of features able to classify a known case. Two feature sets are proposed, one for use on first evaluation and one for dogs with a history of skin problems. A sum for each list when applied to the whole population (including controls) was able to classify 'Cases' with a sensitivity of 89% to 94% and specificity of 71% to 69%, respectively, and identify potentially undiagnosed cases. Our findings demonstrate for the first time that owner questionnaire data can be reliably used to aid in the diagnostic process of cAD.

The ethics and role of AI with fresh and frozen semen in dogs.

The use of artificial insemination (AI) with fresh semen has resulted in many benefits for the management of dog breeding, but there are disadvantages that can sometimes be overlooked. Furthermore, poorer quality semen arising as a result of cryopreservation necessitates uterine insemination, which raises the potential for surgical insemination. A number of significant ethical concerns have been raised by key stakeholders (such as The Kennel Club and the Royal College of Veterinary Surgeons) about AI per se, but particularly about the use of surgical insemination. This paper discusses the technological development of AI and explores a number of the ethical issues raised by its application to dog breeding. An Ethical Matrix method is used to map the potential ethical issues for key interest groups, namely dogs, breeders, owners, veterinarians and wider society. There are national variations in the way in which institutions have evaluated potential ethical impacts, and this is reflected in the different regulatory frameworks governing the use of AI in dogs. In order to facilitate decision-making and reduce some of the ethical risks associated with this technology, the veterinary research community could take several proactive steps including: (i) clarifying clinical decision-making processes, (ii) enhancing informed choice among clients and (iii) increasing the knowledge-base of potential impacts of AI.

The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow.

In cattle, the first postpartum dominant follicle grows slower and produces less oestradiol in animals with high numbers of bacteria contaminating the uterine lumen. However, only bacteria that are uterine pathogens are correlated with severe clinical disease and an increased inflammatory response. It is unknown whether the effect on the ovary in relation to uterine bacterial contamination is associated with the presence of recognised uterine pathogens. Therefore, the present study examined the relationship between pathogenic bacteria in the postpartum uterine lumen, follicle growth and function and the formation of a competent corpus luteum. In addition, peripheral plasma concentrations of immune mediators were quantified. Swabs were collected from the uterine lumen of cattle on day 7 postpartum. Bacteria were cultured and identified and bacterial growth was scored semi-quantitatively. Animals were categorized into high or low recognized uterine pathogen contamination groups based on the number of colonies. Ovarian structures were monitored by daily transrectal ultrasonography and blood samples were collected. In animals with high numbers of uterine pathogens on day 7 postpartum, the diameter of the first postpartum dominant follicle was smaller and plasma oestradiol concentrations were lower. In addition, these animals had smaller corpora lutea, which produced less progesterone. Furthermore, animals with a high day 7 uterine pathogen growth density had higher peripheral concentrations of acute phase proteins. Thus, contamination of the uterus with recognized uterine pathogens is associated with ovarian dysfunction during the postpartum period. Furthermore, infection results in an increase in the production of inflammatory mediators.

Treatment of spontaneous pyometra in 22 bitches with a combination of cabergoline and cloprostenol.

Twenty-two bitches with ultrasonographically diagnosed spontaneous pyometra were treated with a combination of 5 microg/kg cabergoline per day and 5 mug/kg cloprostenol every third day, and potentiated sulphonamide twice a day. Bitches with either open-cervix or closed-cervix pyometra showed a rapid clinical improvement, associated with a reduction in plasma progesterone concentration, increased vulval discharge and a reduction in the diameter of the uterus. The haematological profiles of 21 of the bitches returned to normal within six days of treatment, and their biochemical profiles returned to normal within nine days; 19 of the bitches were managed successfully by a 10-day period of treatment. Two bitches required a further three days of treatment, and in one bitch with a partial uterine torsion the treatment was not successful. Adverse effects of the treatment were limited to the 60 minutes immediately after the administration of prostaglandin, and included retching, vomiting, mild abdominal straining, diarrhoea and panting. The incidence of adverse effects was reduced after each successive dose of prostaglandin. Eleven of the 21 successfully treated bitches were mated at the next oestrus, and seven became pregnant; their litters were smaller than the published breed averages. In four of the bitches the pyometra recurred after the next oestrus.

Ultrasonographic characteristics of early pregnancy failure in bitches.

Resorption of conceptuses was identified in 6 of 20 bitches examined daily from 15 to 35 d after the estimated pre-ovulatory LH surge. One or more conceptuses were resorbed in each bitch but there was continuation of the remaining pregnancy to term. This paper details the ultrasonographic embryological features of normal and resorbing conceptuses. Impending resorption may be predicted by detecting a delay in the time of development of a specific embryological feature or measuring a slow growth rate. Interestingly, there were greater number of conceptuses initially identified in bitches that subsequently resorbed than in those which did not.

Relationship between the fertile period and sperm transport in the bitch.

Following ejaculation into the vagina, dog sperm remain functionally competent for many days, acquire the ability to fertilize, and are delivered to an appropriate site within the uterine tube synchronously with the appearance of fertile oocytes. The mechanisms involved in regulating this system are complex, and allow for sperm storage within the female reproductive tract. The aim of this paper is to review current knowledge of the transportation of sperm and their biology within the reproductive tract of the bitch.

Effects of coitus and the artificial insemination of different volumes of fresh semen on uterine contractions in mares.

Uterine contractions may play an important role in the transportation of spermatozoa towards the site of fertilisation in the oviduct of mares. M-mode ultrasound was used to measure the number, amplitude and duration of uterine contractions in each uterine horn and the uterine body of oestrous mares for four minutes before and four minutes after either coitus, or the artificial insemination of either 80.0 ml of fresh semen or 10.0 ml of fresh semen. The direction of the uterine contractions in each uterine horn and the uterine body was measured before and after coitus. Coitus and the insemination of 80.0 ml semen significantly increased the total number, mean amplitude and mean duration of contractions in all parts of the uterus. The insemination of 10.0 ml of semen did not affect the total number or the mean duration of contractions in the uterine horns. Their mean amplitude was increased, but largely owing to the results from one mare; it also did not affect the contractions in the uterine body. There was no significant difference between the percentage of contractions moving in a cervicotubal or tubocervical direction after coitus in any part of the uterus examined.

Incidence and impact of dog attacks on guide dogs in the UK: an update.

Data on dog attacks on Guide Dogs' stock were reviewed to investigate the characteristics of the attacks. An average of 11.2 attacks occurred each month. Nearly all of the attacks occurred in public areas, 68.4 per cent of victim dogs were qualified guide dogs and 55.5 per cent of victim dogs were working in harness when they were attacked. Guide Dogs' stock were injured in 43.2 per cent of attacks and veterinary costs for attacks were estimated at £34,514.30. Over 40 per cent of qualified guide dogs' working ability was affected and >20 per cent of qualified guide dogs required some time off from working after a dog attack. Twenty dogs were permanently withdrawn from the Guide Dogs' programme as a result of dog attacks, 13 of which were qualified and working with guide dog owners at the time of the withdrawal; this resulted in a financial cost of >£600,000 to the charity. More importantly perhaps, temporary and permanent withdrawals have a significant impact upon the mobility and independence of guide dog owners and in many cases significantly impacted their emotional well-being.

Using the incidence and impact of health conditions in guide dogs to investigate healthy ageing in working dogs.

This study aimed to use retirement data from working guide dogs to investigate healthy ageing in dogs and the demographic factors that influence ageing. Using a dataset of 7686 dogs spanning 20 years, dogs withdrawn for health reasons before they reached retirement were identified. Cases of retirement for old age, rather than for health reasons, were also recorded, as was the length of working life for all dogs. Specific health reasons were grouped into 14 different health categories. The influence of purebred or crossbreed, breed, and sex on the incidence of these health categories and the length of working life within each health category was considered. The majority (n = 6465/7686; 84%) of working guide dogs were able to function as guide dogs until they had worked for 8.5 years, when they retired. This working life might constitute a reference for the different breeds considered, with the exception of the German shepherd dog, which had a shorter working life. The most common reason for health withdrawals was musculoskeletal conditions (n = 387/1362; 28%), mostly arthritis. Skin conditions (mostly comprised of cases of atopic dermatitis) reduced working life most commonly (mean, approximately 5 years). Nervous sensory conditions (35% of which were cases of epilepsy) reduced working life by 3 years.

Veterinary practitioners' selection of diagnostic tests for the primary evaluation of colic in the horse.

The aim of this study was to survey veterinary practitioners' selection of diagnostic tests for horses with clinical signs of abdominal pain. A questionnaire was distributed to veterinary surgeons involved in the primary evaluation of horses with abdominal pain, including the respondent's demographics, selection of diagnostic tests and factors affecting decision-making. Data analysis included descriptive analysis, categorisation of free text and simple univariable correlations to explore the relationships between independent variables and the relative self-estimated frequency that diagnostic tests were performed. A total of 228 responses were analysed. Participants worked in mixed practice (55.7 per cent), first opinion equine (22.8 per cent), first and second opinion equine (17.9 per cent) and referral practice (3.1 per cent). The majority (48.2 per cent, 105/218) were very confident managing a colic case (confidence level 4/5). The most frequently used diagnostic tests were 'response to analgesia' (87.2±24.0 per cent cases), rectal examination (75.9±21.2 per cent) and nasogastric intubation (43.8±27.6 per cent). Approach varied between practitioners, and for all diagnostic tests with frequency of use ranging from 0 to 100 per cent of cases. 'Risk to personal safety' was the most common reason for not using rectal examination. Practitioner's opinion of their confidence level in managing a colic case was associated with how frequently they used different diagnostic tests. There was marked variation in practitioners' approaches, highlighting the need for further evidence to support decision-making.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

REASONS FOR PERFORMING STUDY: Colic is the most common equine emergency problem, and one of the main causes of equid deaths. The horse owners' ability to recognise colic is a critical first step in determining case outcome. OBJECTIVES: To assess equine owners' knowledge and recognition of colic. STUDY DESIGN: Online questionnaire of horse owners. METHODS: An online survey was designed to evaluate owners' approach to colic in the horse. The survey included questions on owner demographics, their recognition of colic (including owner's opinions of their ability to recognise colic, their approach, and their recognition of colic using case vignettes), and their knowledge of normal ranges for clinical parameters. Descriptive and chi squared statistical analysis was performed. RESULTS: The survey was completed by 1061 UK respondents. Six per cent of owners thought they could recognise all types of colic, 61% said they could recognise most cases and 30% said they could recognise some but not all cases. Owners said they would assess faecal output (73% of respondents), gastrointestinal sounds (69%), respiratory rate (62%) and heart rate (50%) in horses with suspected colic. One fifth (22%) of owners would call a vet immediately without assessing any parameters. Many respondents either did not know, or provided incorrect estimates of normal values for clinical parameters: 30.4% were 'unsure' of the normal heart rate and 35.5% gave heart rate values which were outside reference ranges; only 24.5% gave appropriate values for normal respiratory rates and only 31% gave normal temperature values. There was no statistical significance between participants' age, educational qualifications, or their experience with horses and their knowledge of normal clinical parameters. CONCLUSIONS: Owners varied in their approach and ability to recognise colic, and many had significant gaps in their knowledge of normal parameters. Educational materials and/or training to assist owners could help address these issues. Ethical animal research: The study did not involve animal research. The study was reviewed and approved by the Ethics Committee, School of Veterinary Medicine and Science, University of Nottingham. The questionnaire was conducted in accordance with the 1998 Data Protection Act, and the British Educational Research Association's Revised Ethical Guidelines for Educational Research (2004). SOURCE OF FUNDING: Adelle Bowden's studentship is funded by the School of Veterinary Medicine and Science, University of Nottingham. Competing interests: None declared.

Hazardous air pollutant emissions from gas-fired combustion sources: emissions and the effects of design and fuel type.

Air emissions from gas-fired combustion devices such as boilers, process heaters, gas turbines and stationary reciprocating engines contain hazardous air pollutants (HAPs) subjected to consideration under the federal clean air act (CAA). This work presents a recently completed major research project to develop an understanding of HAP emissions from gas-fired boilers and process heaters and new HAP emission factors based on field emission tests of gas-fired external combustion devices used in the petroleum industry. The effect of combustion system design and operating parameters on HAP emissions determined by both field and research tests are discussed. Data from field tests of gas-fired petroleum industry boilers and heaters generally show very low emission levels of organic HAPs. A comparison of the emission data for boilers and process heaters, including units with and without various forms of NOx emission controls, showed no significant difference in organic HAP emission characteristics due to process or burner design. This conclusion is also supported by the results of research tests with different burner designs. Based on field tests of units fired with natural gas and various petroleum industry process gases and research tests in which gas composition was intentionally varied, organic HAP emissions were not determined to be significantly affected by the gas composition. Research data indicate that elevated organic HAP emission levels are found only under extreme operating conditions (starved air or high excess air combustion) associated with poor combustion.

Synthetic oviductal fluid and oviductal cell coculture for canine oocyte maturation in vitro.

The perfection of in vitro maturation in the bitch has yet to be achieved, and is an essential prerequisite for gamete salvage programmes in endangered canine species. In contrast to most mammals, the bitch ovulates an immature oocyte which undergoes meiotic maturation within the oviduct. A model of the oviductal environment may therefore be useful for performing in vitro maturation. This study was performed to investigate the effect of introducing an oviductal element to the culture environment, first with the use of a synthetic oviductal fluid (SOF), and secondly, using coculture with isolated canine oviductal epithelial cells, upon the rate of oocyte maturation in vitro. It was found that there was no difference in the proportion of oocytes undergoing germinal vesicle breakdown (GVBD) after 48 h in culture between SOF containing 0.3% bovine serum albumin (BSA, 45%), containing 4% BSA (36%) and control medium 199 (27%). There was also no difference in oocyte nuclear maturation to metaphase I/anaphase I/metaphase II (MI/AI/MII) after 48 h in culture between SOF containing 0.3% BSA (5%), containing 4% BSA (7%) and control medium 199 (6%). In addition, there was no difference in oocyte nuclear maturation to MI/AI/MII after 96 h between SOF containing 0.3% BSA (0), containing 4% BSA (7%) and control medium 199 (11%). In contrast, the proportion of oocytes undergoing GVBD after 96 h in culture was affected by the treatment used, with 27% in SOF + 0.3% BSA, 62% in SOF + 4% BSA and 63% in medium 199. It was found that there was no difference in the proportion of oocytes undergoing GVBD between the coculture treatments 199 (33%), 199 + cells (37%), coculture medium (30%) and coculture medium + cells (49%), and for oocyte nuclear maturation to MI/AI/MII, between medium 199 (2%), 199 + cells (0), coculture medium (6%) and coculture medium + cells (2%) after 48 h in culture. In addition, there was no difference in oocyte nuclear maturation to GVBD after 96 h between 199 (61%), 199 + cells (59%), coculture medium (65%) and coculture medium + cells (53%). In contrast, the proportion of oocytes maturing to MI/AI/MII after 96 h in culture was affected by the treatment used, with a significant difference between 199 (0), 199 + cells (9%), coculture medium (0) and coculture medium + cells (0). It was shown, therefore, that the culture of oocytes in the SOF improved oocyte nuclear maturation when supplemented with a high concentration of protein and that culture in the presence of oviductal epithelial cells improved oocyte maturation, but only after a prolonged period of time.