Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation     

WMC Maxwell  I Parrilla  I Caballero  E Garcia  J Roca  EA Martinez  JM Vazquez  D Rath 《Reproduction in domestic animals》2007,42(5):489-494

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.  相似文献   

Plasma aldosterone, vasopressin and atrial natriuretic peptide in hypovolaemia: a preliminary comparative study of neonatal and mature horses   总被引：1，自引：0，他引：1  

Hollis AR  Boston RC  Corley KT 《Equine veterinary journal》2008,40(1):64-69

REASON FOR PERFORMING STUDY: Neonatal foals succumb rapidly to hypovolaemic shock in comparison to mature horses; they do not consistently increase their heart rate in response to hypotension and respond differently to fluid administration. The hormonal responses to hypovolaemia in the horse and foal require investigation. HYPOTHESIS: The hormonal responses to hypovolaemia and fluid administration differ between mature and neonatal horses. METHODS: Five mature horses and 5 neonatal foals fulfilling predetermined criteria for hypovolaemia, were included in the study. A blood sample was taken at admission and after normalisation of fluid balance. These were analysed for plasma aldosterone, vasopressin (AVP) and atrial natriuretic peptide (ANP). Normally distributed variables were compared using the Student's t test and nonparametric data using the Mann-Whitney U test. RESULTS: ANP, AVP and aldosterone were higher before fluid resuscitation than after fluid resuscitation in mature horses. Aldosterone was higher before than after fluid resuscitation in foals, and was higher in foals both before and after fluid resuscitation than in mature horses. ANP was lower in mature horses after fluid resuscitation than in foals. No other comparisons were significantly different. CONCLUSIONS: The hormonal responses of the mature and neonatal horses are different during hypovolaemia and following fluid resuscitation. POTENTIAL RELEVANCE: The differences in the hormonal responses to hypovolaemia and fluid resuscitation may be important when considering fluid resuscitation of hypovolaemic horses and foals, and warrants further investigation.  相似文献   

Low-Dose Insemination in Pigs: Problems and Possibilities     

JM Vazquez  J Roca  MA Gil  C Cuello  I Parrilla  I Caballero  JL Vazquez  EA Martínez 《Reproduction in domestic animals》2008,43(S2):347-354

Low-dose AI procedures are required by the pig industry to efficiently utilize emerging sperm technologies, such as cryopreservation and sex-sorting. Currently, several different procedures for inseminating with a low or very low number of spermatozoa have been described. Deep intrauterine insemination allows the deposition of the spermatozoa in the depth of the uterine horn, allowing a significant reduction in the number of spermatozoa inseminated with maintenance of optimal reproductive performance. Intra-oviductal laparoscopic insemination has been recently applied in pigs. This technique has proved to be applicable with diluted and sex-sorted spermatozoa. This review discusses several problems encountered during the development of deep intrauterine insemination and intra-oviductal laparoscopic insemination of pigs and provides potential solutions for the practical application of both the technologies.  相似文献   

Cardiac output determination by use of lithium dilution during exercise in horses     

Durando MM  Corley KT  Boston RC  Birks EK 《American journal of veterinary research》2008,69(8):1054-1060

OBJECTIVE: To compare cardiac output (CO) obtained by the lithium dilution method (LiDCO) with CO calculated from the Fick principle (FickCO), in horses maximally exercising on a high-speed treadmill. ANIMALS: 13 Thoroughbreds. PROCEDURES: In part 1 of the study, 5 horses performed a warm-up (walk, trot, and canter) and exercise test (walk, trot, canter, and gallop [90% to 100% maximum oxygen consumption [{[Formula: see text]O(2)max}]) with measurements of LiDCO and FickCO obtained simultaneously after 60 seconds at each exercise level, for a total of 7 measurements. In part 2 of the study, 8 horses performed a warm-up (walk, trot, and canter) followed by an exercise test (walk and gallop [90% to 100% [Formula: see text]O(2)max], repeated twice). Measurements of LiDCO and FickCO were obtained 60 seconds into the first walk and each gallop of the exercise tests, for a total of 3 measurements. RESULTS: Cardiac output increased significantly with increasing speeds by use of both methods. In part 1, lithium dilution significantly overestimated CO, compared with the Fick principle, during the exercise test (as both injection number and exercise intensity increased). Mean +/- SD bias was 246 +/- 264 mL of blood/min/kg in part 1 and 67 +/- 100mL of blood/kg/min in part 2. Three injections of lithium (part 2) did not result in the same degree of overestimation of LiDCO that was observed with 7 injections (part 1). CONCLUSIONS AND CLINICAL RELEVANCE: Lithium dilution may be an acceptable substitute for the Fick principle as a means to measure CO in maximally exercising client-owned horses.  相似文献   

Importance of oil overlay for production of porcine embryos in vitro          下载免费PDF全文

CA Martinez  EA Martinez  MA Gil 《Reproduction in domestic animals》2018,53(2):281-286

Technologies to edit the zygote genome have revolutionized biomedical research not only for the creation of animal models for the study of human disease but also for the generation of functional human cells and tissues through interspecies blastocyst complementation technology. The pig is the ideal species for these purposes due to its great similarity in anatomy and physiology to humans. Emerging biotechnologies require the use of oocytes and/or embryos of good quality, which might be obtained using in vitro production (IVP) techniques. However, the current porcine embryo IVP systems are still suboptimal and result in low monospermic fertilization and blastocyst formation rates and poor embryo quality. During recent years, intensive investigations have been performed to evaluate the influence of specific compounds on gametes and embryos and to avoid the use of undefined supplements (serum and serum derivate) in the incubation media. However, little consideration has been given to the use of the mineral oil (MO) to overlay incubation droplets, which, albeit being a routine component of the IVP systems, is a totally undefined and thus problematic product for the safety of gametes and embryos. In this review, we provide an overview on the advantages and disadvantages of using MO to cover the incubation media. We also review one important concern in IVP laboratories: the use of oils containing undetected contamination. Finally, we discuss the effects of different types of oils on the in vitro embryo production outcomes and the transfer of compounds from oil into the culture media.  相似文献   

The Effects of Hoechst 33342 Staining and the Male Sample Donor on the Sorting Efficiency of Canine Spermatozoa     

C Rodenas  X Lucas  T Tarantini  D Del Olmo  J Roca  JM Vazquez  EA Martinez  I Parrilla 《Reproduction in domestic animals》2014,49(1):115-121

The aim of this study was to evaluate the influence of Hoechst 33342 (H‐42) concentration and of the male donor on the efficiency of sex‐sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 10⁶ sperm/ml, split into four aliquots, stained with increasing H‐42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non‐viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X‐ and Y‐chromosome‐bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H‐42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H‐42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex‐sorting of canine spermatozoa by flow cytometry can be performed successfully using H‐42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives.  相似文献   

The variation of relative water content,SPAD chlorophyll meter reading,stomatal conductance,leaf area,and specific leaf area of Jerusalem artichoke genotypes under different durations of terminal drought in tropical region     

Aunchana Chaimala  Sanun Jogloy  Nimitr Vorasoot  Corley C. Holbrook  Craig K. Kvien  Suwit Laohasiriwong 《Journal of Agronomy and Crop Science》2023,209(1):12-26

This work assessed the responses of Jerusalem artichoke (JA) genotypes experienced to different durations of terminal drought for relative water content (RWC), SPAD chlorophyll meter reading (SCMR), stomatal conductance (SC), leaf area (LA) and specific leaf area (SLA), investigated the significant contributor of physiological traits to yield and yield reduction and diversity of physiological traits in terminal drought tolerance genotypes. Field research was conducted during the late-rainy season for 2 years using a split-plot design with four replications. The three irrigation treatments involved no-drought (SD0), short (SD1) and long (SD2) durations of terminal drought were arranged as main plots, and sub-plots were six genotypes of JA. The current study revealed that RWC, SC, LA, and SLA, were drastically reduced whilst SCMR values slightly increased under SD1 and SD2 compared to SD0. According to multiple linear regression indicated that SLA (SD0), and RWC (SD1 and SD2) had high contributions to tuber fresh weight (TFW), whereas SC had a high contribution to TFW reduction under terminal drought. Principal component analysis also confirmed that the diversity of LA, RWC, and SLA had maximized contributing traits, followed by HI, SCMR and SC in these breeding materials of JA under three irrigation treatments. Our finding emphasized that JA125 (low SLA), HEL256 and JA37 (high SLA) lead to the high performance of TFW under SD0, and they exhibited high TFW under SD1 by performing medium RWC. Likewise, JA4 and JA37 had high TFW under SD2 caused by medium RWC. HEL253 and JA60 were identified as terminal drought-tolerant genotypes because of performed medium SC, which contributed to a low reduction in TFW. Our promising results are the basis for further studies, SLA, RWC and SC are an important keys for screening drought tolerance (low economic yield reduction) and these genotypes studied could be used for parental lines to improved drought tolerance progenies through breeding programs under different durations of terminal drought-prone areas.  相似文献