Fertilizing ability of canine spermatozoa cryopreserved with skim milk‐based extender in a retrospective study

We previously reported that skim milk (SM) is an effective cryoprotectant for cryopreservation of canine spermatozoa instead of egg yolk (EY), which is the conventional cryoprotectant. In this study, the fertilizing ability and practical use of frozen canine spermatozoa prepared with SM were evaluated by transcervical insemination. Frozen‐thawed spermatozoa were inseminated one to four times on days 2–9 after the LH surge. In SM group, a single transcervical insemination (TCI) on Day 5 led to higher delivery rate (83%) than any other days (33%–50%) post‐LH surge. In EY group, delivery rate in double TCI on days 5 and 6 (71%) was higher compared to any other experimental groups (0%–44%). Regardless of single or double, TCI on Day 5 or Day 6 led to higher litter sizes in SM or EY groups, respectively. The breeding efficiency and litter size of single TCI on Day 5 (4.2) and double TCI on Day 5 and Day 6 (3.7) were significantly higher than in the other experimental groups in SM and EY groups, respectively (p < .05). These findings suggest that skim milk is a suitable alternative to egg yolk for cryopreservation of canine spermatozoa, and the suitable timing for insemination might be on Day 5 post‐LH surge.     

A comparison of liquid and lyophilized egg yolk plasma to low density lipoproteins for freezing of canine spermatozoa

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low‐density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10 min after thawing using computer‐assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p > 0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo‐osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC‐Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10 min after thawing. Both liquid and lyophilized 40% EYP‐based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen.     

Improved conception rates in sows inseminated with cryopreserved boar spermatozoa prepared with a more optimal combination of osmolality and glycerol in the freezing extender

Cryoprotectant agents (CPAs) are added in freezing extenders to prevent intracellular ice crystal formation. However, it has been reported that high dose of CPAs confer toxicity on spermatozoa. Recently, the reduction of intracellular water by a high osmolality solution has also resulted in the suppression of ice crystal formation in spermatozoa, suggesting that the optimal combination of glycerol concentration and freezing extender osmolality could contribute to the development of effective sperm cryopreservation techniques. In this study, we investigated the motility, membrane and acrosomal integrity of frozen-thawed boar spermatozoa treated with freezing extender (NSF) of varying osmolalities (300, 400, 500 mOsm/kg) and final concentrations of glycerol (0.5, 1, 2, 3%). The spermatozoa that were treated at 400 mOsm/kg and 2% glycerol showed significantly higher rates of motility and membrane integrity compared with those in other treatment groups. In addition, the conception and implantation rates of swine artificially inseminated with spermatozoa frozen by the novel freezing extender (conception; 79%, implantation; 57.5%) were significantly higher than those of frozen-thawed spermatozoa treated in the conventional NSF (300 mOsm/kg, 3% glycerol) (conception; 29%, implantation; 33.8%). From these results, we concluded that the novel hyperosmotic (400 mOsm/kg) and low-glycerol (final concentration 2%) freezing extender is beneficial for the cryopreservation of boar spermatozoa.     

Effect of cooling rate and cryoprotectant on the cryosurvival of equine spermatozoa

Cryosurvival of cells is reduced if the cooling rate used is suboptimal. If cells cool too rapidly, intracellular water will freeze, causing intracellular ice crystals. However, if spermatozoa are cooled too slowly, excessive cellular dehydration occurs, causing irreversible damage to cellular compartments. In addition, cryoprotectants are added to the freezing diluent to protect cells from damage during cryopreservation. This study was conducted to determine the optimal cooling rate for stallion spermatozoa frozen in the presence of three different cryoprotectants. Spermatozoa were frozen in a skim milk, egg yolk diluent containing 4% glycerol, and ethylene glycol or dimethyl formamide at 10 different cooling rates ranging from 5°C/min to 50°C/min. The percentage of viable spermatozoa was higher for spermatozoa cooled at 10°C/min than at 50°C/min (P < .05). Spermatozoa frozen using glycerol as the cryoprotectant had higher percentages of motile and progressively motile spermatozoa compared with spermatozoa frozen using the other two cryoprotectants (P < .05). In conclusion, the cryosurvival of stallion spermatozoa is similar when cooling rates of 5°C/min to 45°C/min are used, and when 4% cryoprotectant is used, glycerol is a more effective cryoprotectant than ethylene glycol or dimethyl formamide.     

Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen–thawed Thai native bull sperm

This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen–thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen–thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post‐thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post‐thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI.     

Hemato-biochemical changes, disease incidence and live weight gain in individual versus group reared calves fed on different levels of milk and skim milk

A 2 × 3 factorial design was used to study the impact of rearing systems, individual (I) versus group (G) and different levels of milk/skim milk feeding (F1, F2 and F3) on hemato‐biochemical profile, disease incidence and average daily gain of crossbred (Bos indicus × Bos taurus) calves. Six calves were taken in each group on the basis of their birth weight and housed in individual (2.20 × 1.16 m²/calf) or in group pens (2.20 × 1.03 m²/calf). After 3 days of colostrum feeding, calves were allocated to one of three different milk feeding schedules: milk fed up to 8 weeks of age (F1), milk up to 4 weeks followed by 50% replacement by skim milk up to 6 weeks and 100% thereafter (F2) and 100% replacement of milk with skim milk after 4 weeks (F3). Calf starter and cereal green fodders were fed ad libitum from the second week of age and continued for 14 weeks. Parameters on health and disease profiles of calves (disease incidence, duration of illness, response to treatment and recovery) and weekly live weight change were recorded. Calf scour predominated (52.8%), followed by joint ill (25.0%) and respiratory infections (19.4%). The disease incidence was greater (P < 0.01) in individually housed calves (94.4 vs. 55.9%). The management of navel ill required longer recovery (7.01 days) followed by joint ill (4.87 days) and respiratory infection (4.86 days). The average daily gain during 0–14 weeks of age was higher (P < 0.01) in group‐housed calves (433 ± 22 vs. 355 ± 31 g), while the effect of feeding was not significant. Blood samples collected at 4, 8 and 14 weeks of age showed some periodic higher concentrations (but within normal range) of plasma urea and total protein in group housed calves on F2 and F3 feeding schedules in response to high protein intake. Other parameters remained non‐significantly different. Thus, group‐housed calves can be reared successfully with comparatively better performance and less illness than individually housed ones under the present health care and housing management system. However, the system should not be used as a substitute for good management, and frequent observations of calves should be an integral part of any successful rearing program.     

Dark adaptation time in canine electroretinography using a contact lens electrode with a built-in light source

The purpose of this study was to evaluate the dark adaptation time in canine electroretinography (ERG) using a contact lens electrode with a built-in LED. Twelve eyes of six normal laboratory beagle dogs were used and exposed to steady room light at 500 lux for 30 min for light adaption. ERG was recorded at different time points during dark adaptation in sedated and light-adapted beagles. The stimulus intensity was 0.0096 cd/m²/sec. The b-wave amplitude increased significantly until 25 min of dark adaptation, whereas no significant changes in amplitudes were observed after 30 min. Dark adaptation for more than 25 min would be necessary for accurate ERG in canine ERG using a contact lens electrode with a built-in LED.     

Cryopreservation of llama semen using a combination of permeable cryoprotectants

Semen cryopreservation is not available for massive use in South American Camelids (SACs) due to the lack of an efficient protocol and the low pregnancy rates obtained with artificial insemination (AI). The use of a single cryoprotectant (CP) is commonly used in SACs frozen semen. The objective of the study was to evaluate the combined cryoprotective capacity of two permeable CPs at different stages of the cryopreservation protocol in llama semen. Sixteen ejaculates from 4 llama males were analysed, and sperm quality was assayed in raw semen, at 5°C, after equilibration of samples with the CPs and when samples were thawed. The following CPs and combination were used: 6% glycerol (GL), 6% dimethylformamide (DMF) and the combination of both CPs: 3% GL and 3% DMF. A Kruskal–Wallis test and an experimental factorial design, considering one factor with four levels (raw semen, 6% GL, 6% DMF and GL/DMF), were used. Total sperm motility and live sperm with intact acrosomes remained unchanged after equilibration of samples (p > .05). A significant decrease in the percentage of functional membrane, motile and live sperm with intact acrosomes was observed when samples were thawed (GL, DMF and GL/DMF). Nevertheless, the cryopreservation protocols used preserved sperm DNA quality; thus, sperm chromatin condensation and DNA fragmentation were unaffected (p > .05) when GL, DMF and GL/DMF were used. To conclude, no superiority was found between the use of a single or a combination of permeable cryoprotectants to freeze llama semen.     

Detection of intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) and lipid peroxidation during cryopreservation of alpaca spermatozoa

The objective of this study was to detect changes in intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) production and lipid peroxidation during cryopreservation of alpaca spermatozoa. Twelve alpaca semen samples were conventionally cryopreserved. Intracellular superoxide anion and hydrogen peroxide were evaluated by fluorescence microscopy using dihydroethidium (DHE)/YO‐PRO‐1 and dichlorofluorescein diacetate (H2DCFDA)/propidium iodide (PI), respectively. Evaluations were performed during cooling curve at (1) 25°C, (2) 15°C, (3) 5°C/0 min, (4) 5°C/15 min, (5) 5°C/30 min and (6) after freezing/thawing. Evaluation of lipid peroxidation by measuring malondialdehyde (MDA) was performed at 25°C, 5°C/30 min and after thawing. Maximum percentages of total spermatozoa producing superoxide anion and hydrogen peroxide were found at 5°C/30 min (62.8 ± 6.3% and 30.5 ± 5.6%, respectively), and these results were higher (p < .05) than initial (25°C: 10.8 ± 3.8% and 6.8 ± 0.7%, respectively) and after thawing (29.8 ± 9.5% and 7.5 ± 1.8%, respectively) values. However, considering only viable spermatozoa, production of superoxide anion and hydrogen peroxide during overall stabilization at 5°C (>76% and >91%, respectively) and after thawing (74.9 ± 5.0% and 78.9 ± 2.2%, respectively) was higher (p < .05) than initial values at 25°C (38.7 ± 3.1% and 53.6 ± 2.0%, respectively). Lipid peroxidation at 25°C, 5°C/30 min, and post‐thawing were 346.5 ± 99.8, 401.1 ± 64.8 and 527.7 ± 142.8 ng/ml MDA, respectively. These results showed that high percentage of viable alpaca spermatozoa produces intracellular reactive species oxygen (ROS) during the cryopreservation process of alpaca semen.     

Cryopreservation of boar semen. III: Ultrastructure of boar spermatozoa frozen ultra-rapidly at various stages of conventional freezing and thawing.

Ejaculated boar spermatozoa subjected to a conventional freezing and thawing process, were ultra-rapidly fixed, freeze-substituted and examined by electron microscopy to monitor the presence of real or potential intracellular ice and the degree of cell protection attained with the different extenders used during the process. Numerous ice crystal marks representing the degree of hydration of the cells were located in the perinuclear space of those spermatozoa not in proper contact with the extender containing glycerol (i.e. prior to freezing). The spermatozoa which were in proper contact with the extenders presented a high degree of preservation of the acrosomes, plasma membranes as well as the nuclear envelopes. No ice marks were detected in acrosomes before thawing, indicating that the conventional assayed cryopreservation method provided a good protection against cryoinjury. The presence of acrosomal changes (internal vesiculization, hydration and swelling) in thawed samples however, raises serious questions about the thawing procedure employed.     

Initial cooling time before freezing affects post‐thaw quality and reproductive performance of rabbit semen

The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post‐thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris‐citrate‐glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post‐thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

Antioxidant effects of supplementation of 3,4-dihydroxyphenyl glycol on sperm parameters and oxidative markers following cryopreservation in canine semen

Supplementing the extender with antioxidants with low molecular weight can enhance the quality of the post-thaw sperm during the freezing process. This study was aimed at determining the impacts of 3,4-dihydroxyphenyl glycol (DHPG) on the spermatozoa of the canine undergoing freeze-thawing process. In this study, 24 ejaculates were obtained from three mixed-breed dogs and were diluted in a Tris-based extender. The diluted semen was divided into aliquots for supplementation of 10, 30, 50 and 70 µg/ml of DHPG, control (without antioxidant) and control sham (DMSO). After being extended, the semen was equilibrated at a temperature of 4°C and then transferred to the straws and kept 4 cm above the liquid nitrogen for 20 min and was finally immersed in the liquid nitrogen. They were cryopreserved for seven days; then, sperm parameters including sperm motility evaluation, motility characteristics, viability, DNA and plasma membrane integrity, total antioxidant capacity (TAC), reduced glutathione content (GSH), antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT] and glutathione peroxidase [GPx]) activity malondialdehyde (MDA) levels were evaluated. This study showed that spermatozoa cryopreservation with 50, 30 and 70 µg/ml of DHPG concentrations had better progressive motility, Curvilinear Velocity, Linearity, viability, intact plasma membrane and the levels of TAC, GPx and GSH were higher than the control group. The 50, 30 and 70 µg/ml of DHPG concentrations led to the significant decrease of DNA damage compared to the control group. Total motility, average path velocity, straight-line velocity and CAT activity were significantly improved in 30 and 50 µg/ml of DHPG concentrations, compared to the control group. Also, the 50 and 30 µg/ml of DHPG concentrations, decreased MDA levels compared to the other groups, significantly. In conclusion, our study showed that the addition 50 µg/ml of DHPG to the canine semen extender improved the semen characteristics and oxidative markers in the cryopreservation process.     

荷斯坦奶牛耳组织成纤维细胞分离培养及冷冻保存方法研究

试验对荷斯坦奶牛耳组织成纤维细胞的分离、体外培养及5种冷冻保存方法进行了研究。结果表明:用DMEM/F12 20%胎牛血清 100IU/mL双抗的完全培养液进行组织块培养,能获得良好的荷斯坦奶牛耳组织成纤维细胞原代培养物;成纤维细胞混合培养物经TrypsinEDTA(0.25%Trypsin,1mmol/LEDTA.4Na)消化所收集到的细胞主要为成纤维细胞,经2~3代传代,可得到纯化的荷斯坦奶牛耳组织成纤维细胞。纯化培养的成纤维细胞在冷冻保护剂和血清成分相同的条件下,选用5种不同的冷冻保存方法进行冷冻保存,其中使用70%细胞悬液 20%胎牛血清 10%DMSO的冷冻保存悬液,先在4℃预冷平衡0.5h,接着在液氮罐口的气态氮中悬挂4h,然后沉入液氮的方法冻存的细胞,解冻后,经台盼蓝染色后进行细胞活力分析,活细胞率为86.68%;培养48h后细胞贴壁率为86.40%,明显高于其他几种方法。     

Comparison of two dry chemistry analyzers and a wet chemistry analyzer using canine serum

Canine serum was used to compare seven chemistry analytes on two tabletop clinical dry chemistry analyzers, Boehringer's Reflotron and Kodak's Ektachem. Results were compared to those obtained on a wet chemistry reference analyzer, Roche Diagnostic's Cobas Mira. Analytes measured were urea nitrogen (BUN), creatinine, glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol and bilirubin. Nine to 12 canine sera with values in the low, normal, and high range were evaluated. The correlations were acceptable for all comparisons with correlation coefficients greater than 0.98 for all analytes. Regression analysis resulted in significant differences for both tabletop analyzers when compared to the reference analyzer for cholesterol and bilirubin, and for glucose and AST on the Kodak Ektachem. Differences appeared to result from proportional systematic error occurring at high analyte concentrations.     

Measurement of membrane integrity in canine spermatozoa using a fluorescent computer-assisted spermatozoal quantification method after SYBR-14/PI staining compared with manual counting after CFDA/PI staining

The aim of this study was to compare measurements of spermatozoal membrane status in dogs using computer-assisted spermatozoal quantification (CASQ) after staining with SYBR-14 and propidium iodide (PI) with manual counting after CFDA/PI staining. CASQ was performed on fresh (n = 11) and thawed cryopreserved canine semen (n = 91) using (1) a red long-pass (LP) filter on an untreated sample (membrane-disrupted spermatozoa, MDS count) and in a sample with all cellular membranes disrupted (total spermatozoal count, TC), (2) green LP filter for a TC and the red filter for an MDS count and (3) a green short-pass filter to obtain a membrane-intact spermatozoa (MIS) count and the red filter to obtain the MDS count, which were added to give a TC (red–green filter CASQ, n = 50). Spermatozoa were also stained with CFDA/PI, manually examined and classified as MIS or MDS. All measurements were performed in duplicate. The percentage of membrane-intact spermatozoa (MIS) was calculated. The percentage of progressively motile spermatozoa (PMS) was determined subjectively. The data were analysed to measure the agreement between the CASQ and CFDA/PI methods, repeatability of the methods and correlation between the MIS and PMS percentage. Compared with the CFDA/PI method, the agreement of MIS percentage with red filter CASQ was −12% to 34%, green LP filter CASQ −42% to 47% and red–green filter CASQ −23% to 29%. The repeatability of the CFDA/PI and red–green filter CASQ methods were the highest. The MIS and PMS percentages were always correlated (p < .05). Measurement of MIS percentage using red and red–green filter CASQ appeared to be the most reliable automated methods.     

Safety and feasibility of short course pre-operative radiation therapy followed by surgical excision for canine solid tumours

Surgical resection of solid tumours, especially in early stages of disease, remains a cornerstone of cancer treatment in dogs and cats. There are numerous publications that show a strong association between local tumour control and outcome. To achieve local control in some cases radiation therapy and surgery are combined, with radiation therapy being delivered in the neoadjuvant or adjuvant setting. The objective of the study was to report acute toxicity and surgical site complication data in dogs that received a short-course pre-operative (SCPO) radiation therapy protocol, followed by surgical excision for various solid tumours. Medical records were reviewed, and data was analysed retrospectively. Dogs were included if a dermal or subcutaneous solid tumour was treated with SCPO radiation therapy and then was resected on the last day of radiation or 2–3 weeks later. A total of 34 dogs with 35 primary tumours were included. Acute radiation toxicity was diagnosed in 14 sites (40%). VRTOG scores were grade 1 in 50%, grade 2 in 43%, and grade 3 in 7%. Surgical site complications were identified in 17% of dogs with an overall surgical site infection rate of 11%. According to the Clavien-Dindo classification, two dogs required medical intervention (grade 2), 1 dog required surgical intervention under general anaesthesia (grade 3b), and 1 dog died as a result of complications (grade 5). Logistic regression analysis found that anatomic site was significantly associated with complications, where tumours located on the extremity was protective (P = .02; OR 0.06).     

Anterior segment fluorescein angiography of the normal canine eye using a dSLR camera adaptor

Purpose To describe anterior segment fluorescein angiography (ASFA) of the normal canine eye using two different sedation/anesthetic protocols and a digital single lens‐reflex (dSLR) camera adaptor. Methods Dogs free of ocular and systemic disease were used for this study. Dogs received maropitant citrate (1.0 mg/kg SQ) and diphenhydramine (2.0 mg/kg SQ) 20 min prior to butorphanol [n = 6] (0.2 mg/kg IV) or propofol [n = 6] (4 mg/kg IV bolus, 0.2 mg/kg/min CRI). Standard color and red‐free images were obtained prior to administration of 10% sodium fluorescein (20 mg/kg IV). Image acquisition was performed using a dSLR camera (Canon 7D), dSLR camera adaptor, camera lens (Canon EF‐S 60 mm f/2.8 macro), and an accessory flash (Canon 580EXII). Imaging occurred at a rate of 1/s immediately following bolus for a total of 30 s, then at 1, 2, 3, 4, 5, and 10 min. Results Twelve dogs with a combined mean age of 5.1 years and various iris colors were imaged. Arterial, capillary, and venous phases were identified and time sequences recorded. Visibility of the vascular pattern was inversely related to iris pigmentation. Complete masking of blood flow was noted with heavily pigmented irises. Vessel leakage was noted in some eyes. Proper patient positioning and restricted ocular movements were critical in acquiring quality images. No adverse events were noted. Conclusion This study demonstrated that quality high resolution ASFA images were obtainable using a novel dSLR camera adaptor. ASFA of the normal canine eye is limited to irises, which are moderately to poorly pigmented. Use of general anesthesia produced higher quality images and is recommended for ASFA in the dog.     

Measurement of membrane integrity in canine spermatozoa using a fluorescent computer‐assisted spermatozoal quantification method and manual counting after eosin‐nigrosin staining compared with manual counting after CFDA/PI staining

The aim of this study was to compare measurement of spermatozoal membrane status using computer‐assisted spermatozoal quantification (CASQ) and eosin‐nigrosin (EN) staining with manual counting after CFDA/PI staining. Analysis was performed on both fresh and thawed cryopreserved canine semen. Membrane‐disrupted spermatozoa (MDS) were counted using CASQ (n = 311) in an untreated sample and a completely membrane‐disrupted sample, and the percentage of membrane‐intact spermatozoa (MIS) calculated: (Total count ? Untreated sample count) ÷ Total count × 100. Spermatozoa were stained with a one‐step EN stain (n = 501), and then, at least 100 spermatozoa were manually examined under ×1,000 magnification and classified as MDS (stained with eosin) or MIS (non‐stained). Spermatozoa from the same samples were also stained with CFDA/PI, and then, at least 200 spermatozoa were manually examined under ×1,000 magnification and classified as MIS (completely stained by CFDA) or MDS. The percentage of progressively motile spermatozoa (PMS) was determined by both computer‐assisted semen analysis (CASA) and subjective methodologies, and the data were subsequently analysed to measure the agreement between the CASQ and EN methods with the CFDA/PI technique using Bland–Altman methodology. Pearson's correlation was measured between the MIS and PMS percentage samples and correlation coefficients compared. The mean MIS percentage was lower for CASQ and higher for EN than in CFDA/PI for all comparisons. The agreement of MIS percentage between CASQ and CFDA/PI was ?20.2% to 32.0%, and between EN and CFDA/PI was ?32.9% to 14.9%. In all methods, the MIS and PMS percentages were correlated (p < .001). Measurement of CFDA/PI appeared to be the most reliable and accurate method of determining MIS percentage in dogs. Further investigation is required to determine whether the CASQ technique can be improved. Eosin‐nigrosin staining also appeared to be unreliable at MIS <80% and overestimated the MIS percentage.     

Effects of storage time and freezing temperature on clinical chemical parameters from canine serum and heparinized plasma

To assess changes in 24 blood constituents in frozen serum and heparinized plasma, blood samples were drawn from 10 clinically normal German Shepherd army dogs. The storage characteristics of nine enzymes (ALP, ALT, amylase, AST, CK, GGT, GLDH, LDH, lipase), and 15 metabolites and minerals (albumin, bile acids, bilirubin, calcium, cholesterol, creatinine, fructosamine, glucose, magnesium, phosphate, potassium, protein, sodium, triglycerides, urea) were studied. Parallel samples of serum and heparinized plasma were stored for 90 and 240 days at two different storage temperatures, -200 degrees C and -700 degrees C. Sixteen of the 24 analytes (ALP, ALT, amylase, AST, CK, GGT, GLDH, LDH, bile acids, calcium, cholesterol, creatinine, fructosamine, magnesium, phosphate, urea) showed statistically significant (p < 0.05) changes during the storage period related to storage time, storage temperature, and sample type. Seven of the analytes (amylase, GGT, GLDH, LDH, bile acids, fructosamine, magnesium) showed changes of possible clinical importance with mean differences from baseline larger than 20% for the enzymes and 10% for the metabolites and minerals during the storage periods.