国外犬精液冷冻技术的研究进展

从犬的冷冻精液成功受孕产仔以来,犬精液冷冻研究已成为犬繁殖技术的一部分,国外已经详细建立了采精、稀释、冷冻、解冻及精液品质评价的有关参数,并运用于实际生产中,最高受胎率可达90%。相比之下,国内在这方面的研究还不够深入。本文就国外的犬精液冷冻研究进行详细综述,以给我国养犬业的发展提供借鉴。     

Evaluation of platelet additive solution for prolonging storage of functional canine platelet concentrate

Objective

To assess storage lesion development, platelet function, and bacterial growth in canine platelet concentrates (PCs) stored in a platelet additive solution (PAS) or a plasma control at 4°C for 21 days.

Design

Prospective, ex vivo, experimental controlled study.

Setting

University veterinary teaching hospital.

Animals

Ten units of canine PCs collected from blood bank donations.

Interventions

The PCs were separated into 2 bags, 1 containing 100% plasma and the other containing 35% plasma and 65% of a PAS (Plasma-Lyte A), and stored at 4°C for 21 days. At days 0, 7, 14, and 21, PCs were analyzed for the presence of swirling, aggregate formation, platelet counts, platelet indices, glucose, lactate, lactate dehydrogenase, Pvco ₂, Pvo ₂, aggregation via light aggregometry, activation percentages using flow cytometry, and bacterial growth.

Measurements and main results

Cold-stored PCs in both PAS and plasma control maintained mean pH >6.8 and mean lactate <9.0 mmol/L over 21 days, with no difference in glucose utilization. Swirl was maintained in both solutions for most days (76/80 combined total samples), with no difference in aggregate formation between solutions. The Pvco ₂ was higher in plasma on all days (P < 0.001), with no difference in Pvo ₂. Platelet indices did not reflect significant storage lesion development in either solution. Lactate dehydrogenase did not differ between solutions but did increase from day 7 to day 21. Mean maximal aggregation percentage was reduced overall but with no significant difference between solutions. The only observed difference in mean activation percentage between solutions was in PAS on day 7, which was significantly higher than plasma (P < 0.05). No bacterial growth occurred during storage.

Conclusions

Cold storage in PAS and plasma allowed PCs to be stored for up to 21 days with minimal storage lesion development, maintenance of platelet function, limited platelet activation, and no bacterial growth within stored bags.     

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.     

Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels

Background: Many Cavalier King Charles Spaniel (CKCS) dogs are affected by an autosomal recessive dysplasia of platelets resulting in fewer but larger platelets. The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). We hypothesized that CKCS both with and without hereditary macrothrombocytosis would have a normal plateletcrit and that the QBC results would better identify the total circulating volume of platelets in CKSC than methods directly enumerating platelet numbers.
Objectives: The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs.
Methods: Platelet counts were determined in fresh EDTA blood from 27 adult CKCS dogs using the QBC, Sysmex XT-2000iV (optical and impedance), CELL-DYN 3500, blood smear estimate, and manual methods. Sysmex optical platelet counts were reanalyzed following gating to determine the number and percentage of normal- and large-sized platelets in each blood sample.
Results: None of the 27 CKCS dogs had thrombocytopenia (defined as <164 × 10⁹ platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum.
Conclusions: The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass.     

Application of vincristine-loaded platelet therapy in three dogs with refractory immune-mediated thrombocytopenia

Three dogs presented with refractory immune-mediated thrombocytopenia (IMT). All patients failed to respond to prednisone, which is considered a mainstay of immunosuppressive therapy. Vincristine-loaded platelets (VLPs), which act selectively on mononuclear phagocytes,were introduced. After the VLPs were transfused, two dogs responded quickly withimproved clinical signs while the third dogwith recurrent IMT was euthanized due to its deteriorating condition. This case report describesthe efficacy of VLP therapy in refractory IMT patients.     

Evaluation of platelet aggregation using a point-of-care instrument in retired racing Greyhounds

Veterinarians involved in Greyhound rescue have anecdotally observed that 10-15% of Greyhounds bleed profusely after simple surgical procedures. In most patients, platelet counts and hemostasis profiles are normal; therefore, it is possible that these dogs have platelet dysfunction. The PFA-100 is a novel point-of-care platelet function analyzer that has recently been evaluated as a rapid method to assess platelet function in dogs. The objectives of this study were to characterize platelet function in a group of healthy Greyhounds by means of the PFA-100. Blood samples were collected from the jugular vein from 30 healthy Greyhounds. CBC, biochemical profile, PFA-100 assay with collagen/epinephrine (COL-EPI) and collagen/ adenosindiphosphate (COL-ADP), plasma von Willebrand factor antigen concentration (vWF:Ag), and vWF collagen-binding assay (vWF:CBA) were performed. PFA-100 closure times (CTs) with COL/ADP ranged from 63 to 92 seconds (mean +/- SD, 74.7 +/- 7.9 seconds) and with COL/EPI from 87 to 238 seconds (138 +/- 41 seconds); vWF: Ag ranged from 22 to 120% (87.52 +/- 25.5%) and vWF: CBA ranged from 36 to 102% (77.4 +/- 17.3%); and platelet counts ranged from 147 to 265 x 10(9)/L (194.6 +/- 31.64 x 10(9)/L). Greyhound CTs were significantly shorter than CTs in a mixed population of 50 healthy non-Greyhound dogs, in which the COL/ADP CTs ranged from 61 to 172 seconds (mean +/- SD, 87 +/- 21.6 seconds), and the COL/ EPI CTs ranged from 81 to 300 seconds (mean +/- SD, 183 +/- 67.6 seconds; P = 0.005 for COL/ADP CT; P = 0.001 for COL/ EPI CT). Also, platelet counts were significantly lower (P = 0.001) and packed cell volume was significantly higher (P = 0.001) in the Greyhound than in the non-Greyhound group. The PFA-100 is a reproducible method that can be used in the clinical setting to assess platelet function in Greyhounds; however, normal CTs in healthy Greyhounds are shorter than in other breeds. The results obtained in this study will be used to screen for abnormal platelet function in Greyhounds with postoperative bleeding.     

Canine and feline blood transfusions: controversies and recent advances in administration practices

ObjectivesTo discuss and review blood transfusion practices in dogs and cats including collection and storage of blood and administration of products. To report new developments, controversial practices, less conventional blood product administration techniques and where applicable, describe the relevance to anaesthetists and anaesthesia.Databases usedPubMed and Google Scholar using dog, cat, blood transfusion, packed red blood cells and whole blood as keywords.ConclusionsBlood transfusions improve oxygen carrying capacity and the clinical signs of anaemia. However there are numerous potential risks and complications possible with transfusions, which may outweigh their benefits. Storage of blood products has improved considerably over time but whilst extended storage times may improve their availability, a phenomenon known as the storage lesion has been identified which affects erythrocyte viability and survival. Leukoreduction involves removing leukocytes and platelets thereby preventing their release of cytokines and bioactive compounds which also contribute to storage lesions and certain transfusion reactions. Newer transfusion techniques are being explored such as cell salvage in surgical patients and subsequent autologous transfusion. Xenotransfusions, using blood and blood products between different species, provide an alternative to conventional blood products.     

Triggers for prophylactic use of platelet transfusions and optimal platelet dosing in thrombocytopenic dogs and cats.

Prophylactic platelet transfusions are frequently given to human patients with hypoproliferative thrombocytopenia. For several decades, the most common transfusion trigger was 20,000/microL, but the trend is now to use 10,000/microL in the absence of other risk factors for bleeding. This trigger seems to reduce the number of transfusions without increasing the risk of severe bleeding. Most studies involved in establishing platelet transfusion policies have involved patients with acute leukemia, with fewer studies involving patients undergoing hematopoietic stem cell transplantation or aggressive chemotherapy for other cancers and patients with aplastic anemia. In the presence of other risk factors for spontaneous bleeding, 20,000/microL is still considered an appropriate trigger. The trigger for prophylactic transfusion before surgery has not undergone the same recent scrutiny as has the trigger for spontaneous bleeding. The recommendation remains to raise the platelet count to 50,000 to 100,000/microL if possible, although it is recognized that surgery and other invasive procedures have been performed at lower platelet counts without major bleeding. Prophylactic transfusion is not used in disorders of platelet consumption and destruction to prevent spontaneous bleeding but is used before surgery. Because of the comparative lack of experience with platelet transfusion in veterinary medicine, it is difficult to make generalizations for dogs and cats. Using the guidelines established for therapeutic and prophylactic transfusion of human patients is a reasonable starting point, however. A therapeutic transfusion policy is suggested in the veterinary setting provided that the patient can be closely observed for critical bleeding and a prompt transfusion can be given. This policy should ultimately reduce the overall number of platelet transfusions given to hospital patients. If an animal cannot be closely observed or the ability to transfuse on demand is limited, prophylactic transfusion is recommended. The triggers for initiating a platelet transfusion in dogs are extrapolated from human data; these values are lower by 50% for cats. Because of the imprecision of platelet counting at low values, platelet counts must always be interpreted in conjunction with clinical signs of hemorrhage. If platelet-rich plasma or platelet concentrate is available, a dose of 1 platelet unit per 10 kg is recommended, although resources may dictate a smaller dose. This will raise the recipient platelet count by a maximum of about 40,000/microL. Assuming a trigger of 10,000/microL, a transfusion will probably be required approximately every 3 days. It must be remembered that the frequency of platelet transfusions may be greater in the presence of factors accelerating platelet loss or destruction. If fresh whole blood is used, a rule of thumb is to transfuse 10 mL/kg, which will raise the recipient platelet count by a maximum of approximately 10,000/microL. Daily transfusions or transfusions every other day will probably be required.     

Prevalence of low automated platelet counts in cats: comparison with prevalence of thrombocytopenia based on blood smear estimation

Abstract: True thrombocytopenia is uncommon in cats; however, low platelet counts frequently are found using automated cell counters. Although this discrepancy is a well known problem, the prevalence of low automated platelet counts in feline blood samples has not been documented. We retrospectively compared the prevalence of low automated platelet counts with low blood smear-estimated platelet counts in feline blood samples. Results of blood sample analysis from 359 cats during a 1-year period at the University of Glasgow Veterinary Haematology Laboratory were examined. Smear estimates of platelet number were done in those cases in which records did not indicate adequate platelet numbers. Platelet counts obtained with an impedance counter (Minos Vet, Abx Hematologie) were <200×10⁹ cells/L in 256 samples (71%) and <50×10⁹ cells/L in 43 samples (12%). However, based on estimation of platelet numbers from blood smears, only 11 samples (3.1%) had platelet counts of <200×10⁹ cells/L and 9 samples (2.5%) had counts of <50×10⁹ cells/L. Four cats with thrombocytopenia estimated by blood smear evaluation had clinical signs of a bleeding disorder. Disorders associated with thrombocytopenia included neoplasia, cytotoxic chemotherapy, and infectious diseases. There was no evidence that delay due to mailing of samples was associated with lower automated platelet counts than would have been obtained on the day of sampling. The high prevalence of apparent thrombocytopenia in automated platelet counts was attributed to a combination of platelet aggregation and the impedance method of cell differentiation by size. Vigilance and careful examination of blood smears is required to identify the few cats with true thrombocytopenia.