Biochemical and Protein Profile of Alpaca (Vicugna pacos) Uterine Horn Fluid During Early Pregnancy

South American camelids show high embryo loss rate, during the first 60 days of pregnancy. One of the factors which may be related to this situation is that over 98% of the embryos implant in the left uterine horn (LUH) even though both ovaries contribute similarly to ovulation. There is scarce information about the uterine environment of female camelids at any physiological state that could explain the capability of the LUH to attract the embryo and maintain pregnancy. We describe, for the first time, the biochemical and protein profile of uterine fluid (UF), addressing the right and LUH environment in non‐pregnant and pregnant alpacas. Different substrates, electrolytes and metabolites were assayed in both uterine horn fluids. Small changes were observed in glucose and total protein levels, which were more noticeable during pregnancy. In addition, 10 specific proteins were found in the left horn fluid in 5‐week‐pregnant alpacas, and two protein bands were identified in non‐pregnant alpaca right horn fluid. These results would provide basic information for identification of possible markers for pregnancy diagnosis, reproductive diseases and hormone‐treated animals evaluation and hence contributing to improve the pregnancy rate.     

Effect of sperm pretreatment with glutathione and membrane destabilizing agents lysolecithin and Triton X‐100, on the efficiency of bovine intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is an assisted reproduction tool with several applications. Its effectiveness in bovines is lower than that in other species, mainly because of difficulties in the decondensation of the sperm nucleus after injection, and the presence of the acrosome and the plasma membrane which remain intact in this procedure. In this study, we assessed the effect of lysolecithin (LL) and Triton X‐100 (TX), in combination with glutathione (GSH) as sperm pretreatments prior to ICSI. The GSH‐LL and GSH‐TX groups showed 0% of spermatozoa with intact membrane (SYBR 14+/PI), in comparison with the control (63.3%) and GSH (65.7%) groups. The proportions of spermatozoa with damaged acrosome membrane in the GSH‐LL, GSH‐TX, GSH and control groups were 46%, 35.9%, 10.5% and 7.5%, respectively. Sperm chromatin decondensation analysis showed that the groups incubated for 3 hr with GSH presented greater decondensation (p < .05). Although fertilization was improved in all treatment groups evaluated, no differences were observed in the cleavage rate 72 hr after activation in the GSH (73.7%), GSH‐LL (80.2%) and GSH‐TX (77.8%) groups compared to the control (66.3%), neither in the blastocyst rate on day 8 (24.0%, 26.2%, 27.1% and 28.4% for the control, GSH, GSH‐LL and GSH‐TX groups, respectively). No differences were also observed in the total number of cells in all groups. In conclusion, although these sperm treatments promoted nuclear decondensation and induced plasma membrane disruption, these effects were not sufficient to improve bovine embryonic development after ICSI.     

Evaluation of a Burdizzo Castrator for Neutering of Dogs

A Burdizzo castrator was evaluated for the neutering of dogs. Histological and morphological changes of spermatic cells and peripheral serum testosterone after challenge with a GnRH-analogue (gonadorelin) were assessed. There was a control group (G1), a surgically castrated group (G2) and a Burdizzo group (G3) divided in two, G3a receiving two crunches in each spermatic cord and G3b receiving one crunch in each spermatic cord. Sixteen days after application of the Burdizzo blood samples were taken from the dogs at 30 min interval during 2 h; after the second sample the dogs were treated with 1 mug/kg body weight of gonadorelin i.v. The same protocol of gonadorelin challenge was performed in G1 and G2 dogs. The G2 dogs were surgically castrated after the second blood sample, before the gonadorelin treatment, and the G1 dogs after the last blood sample. The excised gonads were examined histologically, and sperm smears were prepared from the caudae epididymidis. The testes and plexus pampiniformis of the G1 and G2 dogs had a normal histological appearance, and they had morphologically normal epididymal sperm cells. In all G3 dogs, there was an acute fibrosis with an inflammatory reaction in the plexus pampiniformis. The testes from the G3a dogs showed diffuse areas of infarction and degeneration of the parenchyma. Similar but less diffuse lesions were seen in group 3b dogs. The deferent ducts from all G3 dogs showed vasitis and/or sperm granulomas. Azoospermia or sperm malformations were observed in the epididymal smears from the G3 dogs. Testosterone concentration in the G1 dogs increased after gonadorelin application (p < 0.0001). The G2 dogs had basal testosterone levels after castration (p < 0.001) and did not respond to gonadorelin. Groups 3a and b showed a slight but non-significant increase in testosterone concentration after gonadorelin challenge, supposedly due to the reduction of testicular blood flow and loss of testicular interstitial tissue.     

Relationship between Prenatal Survival Rate at 70 days of Gestation and Morphometric Parameters of Vagina, Uterus and Placenta in Gilts

Swine uterine capacity affects litter size, and it could be used as a selection parameter of reproductive performance. Although there are some controversial results, evidences show that the catheter penetration length is positively correlated with litter size, and it could be used as a tool for predicting selection methods. The aim of this study was to determine whether there is any association between the prenatal survival rate and placental size at 70 days of gestation, the vaginal length [catheter penetration length during artificial insemination (AI)] and the uterine capacity in a homogeneous group of gilts. Sixty-six commercial-line gilts in pre-pubertal phase had their oestrus induced by hormonal treatment [600 UI of Equine Chorionic Gonadtrophin (eCG) i.m. and after a 72-h period 5 mg of luteinizing hormone (LH) i.m.], but only 40 gilts showed cyclicity after induction. The AI catheter penetration length was tested on these 40 gilts at the moment of AI using a calibrated AI catheter. Four gilts returned to oestrus and the other 36 were killed at around day 69 of pregnancy. The uterine length and weight showed a significant and positive correlation with the prenatal survival rate (p <0.05). The catheter penetration length was unable to predict the conceptus survival rate on 70 days of gestation; however, the uterine size influenced the survival rate positively. The mean placental area was positively correlated with the mean placental weight (p <0.0001), and both with the mean foetal weight (p <0.0001 and p <0.001, respectively). The analysis of the results obtained showed that neither did the catheter penetration length measurement during AI, nor the prenatal survival rate on day 70 of pregnancy predict the uterine capacity, but the uterine and placental size had a significant influence on the prenatal survival and foetus weight, respectively.     

Detection of the Matrix Metalloproteinases MMP‐2 and MMP‐9 and Tissue Inhibitors of Metalloproteinases TIMP‐1 and TIMP‐2 in Llama (Lama glama) Oviduct

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in several reproductive events like oocyte–spermatozoa interaction and semen liquefaction. In order to study their role in the llama oviductal reproductive process, MMP activity in oviductal fluid (OF) was assayed. Considering that llama genome sequences are partially known, a strategy to procure cDNA sequences of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 was designed. Afterwards, their expression patterns in the different llama oviductal segments were assayed. Gelatine zymograms detected 62 and 94 kDa protease activities that matched MMP‐2 and pro‐MMP‐9, respectively. Expression pattern analysis showed that MMP and TIMP mRNAs were present in ampulla, isthmus, utero‐tubal junction (UTJ) and papilla. Altogether, these findings support the argument that MMPs/TIMPs are produced in the oviduct and secreted into the oviductal lumen. Our results encourage further studies to elucidate the role of these proteins in reproductive oviductal events.     

Comparison of sevoflurane and isoflurane anesthetic index in unpremedicated dogs

Anesthetic respiratory effects of sevoflurane (SEVO) were compared with isoflurane (ISO) in unpremedicated dogs. Minimum alveolar concentration (MAC), apneic concentration (AC), and anesthetic index (AI) of SEVO and ISO were determined in eight 1‐year‐old healthy dogs, weighing 19 ± 3 kg (mean ± SEM) in a randomized complete block multiple cross‐over design. Dogs were mask‐induced with either SEVO or ISO in 100% oxygen. Following endotracheal intubation, dogs were instrumented, mechanically ventilated, and MAC was determined using a tail‐clamp method. Next, spontaneous ventilation was re‐established, and anesthetic concentration was increased to determine the AC. Throughout the anesthetic event, heart rate (HR), systolic blood pressure (SAP), mean blood pressure (MAP), diastolic blood pressure (DAP), respiratory rate (RR), end‐tidal carbon dioxide (Pe ′CO₂), and oxyhemoglobin saturation (SpO₂) were recorded at 3‐minute intervals. Following AC determination, AI was calculated as AC/MAC, and dogs were allowed to recover. Each dog was anesthetized four times (twice with ISO and SEVO each) at 1‐week intervals. All data were analyzed using the two‐way anova . Multiple comparisons were performed between ISO and SEVO treatments. Statistical significance was set at p < 0.05. Significant differences were noted between agents for MAC (SEVO, 2.13 ± 0.10%; ISO, 1.38 ± 0.14%; p < 0.0001), AC (SEVO, 7.34 ± 0.13%; ISO, 3.60 ± 0.13%; p < 0.0001), and AI (SEVO, 3.46 ± 0.22; ISO, 2.63 ± 0.14; p = 0.0002). Physiologic parameters were compared between SEVO and ISO at 1MAC, 2MAC, 3MAC, and AC. No differences were noted between SEVO and ISO treatments for cardiovascular parameters (HR, SAP, MAP, DAP). Significant differences were noted, favoring SEVO, for all respiratory parameters (RR, Pe ′CO₂, SpO₂) at increasing MAC multiples. Additionally, regression analysis was conducted for physiologic variable data points. Analysis of Pe ′CO₂ data points demonstrated a significant slope difference of ?6.47 ± 1.02 (B_SEVO ? B_ISO; p < 0.0001; r² = 0.6042) favoring SEVO. While expected dose‐related ventilatory depression was noted for both agents, all the respiratory parameters for SEVO demonstrated less respiratory depression than ISO at equipotent doses. These results indicated that SEVO caused less dose‐dependent ventilatory depression than ISO, having a significantly higher AI and causing less detrimental change in pulmonary parameters at increasing levels of MAC.     

Comparison of femoral and auricular arterial blood pressure monitoring in pigs

Objective To compare arterial blood pressure measurements obtained from the femoral and auricular arteries in anaesthetized pigs. Study design Prospective experimental study. Animals Fifteen female Large White pigs were used weighing 21.3 ± 2.3 kg. Methods The pigs were anaesthetized with tiletamine/zolazepam and xylazine administered intramuscularly, and anaesthesia maintained with isoflurane delivered in oxygen/nitrogen. Arterial oxygen partial pressures were maintained between 11.3 and 13.3 kPa and PaCO₂ between 4.6 and 6.0 kPa. Monitoring included electrocardiogram, capnography and invasive blood pressure. The auricular and femoral arteries were catheterized for continuous systolic (SAP), diastolic (DAP) and mean arterial pressure (MAP) measurements. Measurements were recorded every 15 minutes. Statistical analysis involved a Bland–Altman plot analysis. Results The mean difference ± confidence intervals between the femoral and the auricular arterial diastolic, systolic and mean blood pressure measurements during hypotension were 2 ± 7, 2 ± 5 and 2 ± 5 mmHg respectively. In conditions of normotension mean difference ± confidence intervals, of femoral and auricular arterial blood pressure measurements of diastolic, systolic and mean blood pressure were 4 ± 5, 3 ± 7 and 4 ± 4 mmHg respectively. In conditions of increased arterial blood pressure, mean difference ± confidence intervals, of femoral and auricular arterial blood pressure measurements of diastolic, systolic and mean blood pressure were 4 ± 5, 3 ± 8 and 4 ± 4 mmHg respectively. Conclusion Auricular artery catheterization is easier and quicker to perform. Pressure measurements from the auricular artery compared well with the femoral artery. Clinical relevance We found that auricular arterial blood pressures were similar to femoral arterial values under the conditions of this experiment. We did not test extremes of blood pressure or significant alterations in body temperature.     

Development of a method for the identification, using the polymerase chain reaction, of Aspergillus fumigatus isolated from ostriches

Objective To develop a method for identifying DNA of Aspergillus fumigatus from ostriches, using the polymerase chain reaction (PCR). A fumigatus is the principal causative agent of avian aspergillosis.
Design A biochemical trial.
Sample population Twelve Aspergillus fumigatus isolates and three other Aspergillus species.
Procedure PCR primers that were based on the sequence of the alkaline protease gene from human isolates of A fumigatus were used.
Results We successfully tested the method on ostrich isolates from five states and showed that the test is specific for A fumigatus.
Conclusions In most cases the DNA sequence of A fumigatus isolates from ostriches is similar to that of human isolates. DNA sequences vary significantly among A fumigatus isolates, including those from affected ostriches in the same flock. The genetic variation may be used to trace aspergillus infections in ostrich flocks and determine if the disease is transmitted by contact with infected birds.     

Detection of APAF1 mutation in Holstein cows and mummified foetuses in Japanese dairy herds

Some of the highest genetic merit sires have been shown to harbour recessive mutations affecting fertility, which may spread rapidly in the population through AI. These disorders may result in abortion and decline in pregnancy per insemination in cows. This study was carried out on 240 Holstein‐Friesian cows and 15 mummified foetuses. Blood and tissue samples were collected from the cows and mummified foetuses, respectively, for DNA extraction. Allele‐specific PCR was designed for the detection of the cows and foetuses carrying the nonsense mutation (C/T) in apoptosis peptide activating factor 1 gene (APAF1). The mutant allele frequency of the APAF1 in carrier cows and mummified foetuses was calculated. Milk samples were taken from the carrier and non‐carrier cows for progesterone assay. The allele‐specific PCR reaction efficiently distinguished the C/T mutation in APAF1. Of 240 cows, seven cows (2.9%) were diagnosed to carry one copy of the mutant allele of APAF1. However, the carrier frequency was 33.3% in mummified foetuses (five of 15). The mutant allele frequency was 0.02 and 0.17 in the cows and mummified foetuses, respectively. Concentrations of progesterone did not differ between cows with APAF1 mutation and non‐carrier cows during 45 days post‐insemination. This study provided allele‐specific PCR for the detection of APAF1 mutation in cows. Moreover, it reports the carrier and mutant allele frequencies of APAF1 in dairy cows and mummified foetuses in Japan.