胚胎移植技术的应用及其发展

动物胚胎移植首次成功(Heape 1891)距今已近一百年了。在这期间,又有多种动物胚胎移植试验成功,技术也不断改进。但是,进展较大的还是近二十年间。1981年Betterid-ge总结了胚胎移植历史,列出了13种动物胚胎移植首次成功的时间和试验者[兔1891Heape;大鼠1933 Nicholas;绵羊1934Warwick等;山羊(自身重移植)1934Warwick等;小鼠1942 Fekete＆Lit-tle;奶牛(流产)1949 Umbaugh;山羊1949 Warwick＆Berry;奶牛1951Willett等;猪1951 Kvasnickii;奶牛     

An update on Reproductive Technologies with Potential Short-Term Application in Pig Production

Over the past decade, there has been an increase in the development and/or in the improvement of emerging reproductive technologies in pigs. Among emerging reproductive technologies with potential short-term application in pig production are: artificial insemination with low number of spermatozoa, cryopreservation of spermatozoa and embryos, sperm sexing, and non-surgical embryo transfer. The following review will give emphasis to recent advancements in these reproductive technologies that are starting to show possibilities of serious applications under field conditions.     

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

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.     

Low-Dose Insemination in Pigs: Problems and Possibilities

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.     

Calm Temperament Improves Reproductive Performance of Beef Cows

Profitability of a beef operation is determined by the proportion of cows attaining pregnancy early in the breeding season and those that are pregnant at the end of breeding season. Many factors, including temperament, contribute to those reproductive parameters. The objective of this study was to evaluate effects of temperament on reproductive performance of beef cows. In Experiment 1, Angus and Angus‐cross beef cows (n = 1546) from eight locations were assigned a body condition score (BCS; 1 = emaciated; 9 = obese) and chute exit and gait score (1 = slow exit, walk; calm temperament; 2 = jump, trot or run; excitable temperament). Cows were grouped with bulls (1 : 25 to 1 : 30; with satisfactory breeding potential and free of venereal disease) for an 85‐day breeding season. Pregnancy status and stage of gestation were determined (transrectal palpation) 35 days after the end of the breeding season. Controlling for BCS (p < 0.01) and handling facility (p < 0.0001) and handling facility by temperament score interaction (p < 0.001), breeding season pregnancy rate was lower in excited versus calm cows [88.6% (798/901) vs 94.1% (607/645); p < 0.001]. Cows with an excitable temperament took 24 more days to become pregnant compared to calm cows (median days to pregnancy, 35 vs 59 days; p < 0.0001). In Experiment 2, Angus and Angus‐cross beef cows (n = 1407) from 8 locations were assigned scores for body condition and chute exit and gait (as described in Experiment 1) and assigned to bulls (breeding sound and free of venereal disease; 1 : 25 to 1 : 30) for 85 days. Pregnancy status was determined by transrectal palpation at 2 and 6 months after the onset of the breeding season. Controlling for BCS (p < 0.05), pregnancy loss was higher in excited versus calm cows [5.5% (36/651) vs 3.2% (20/623), p < 0.0001]. In conclusion, beef cows with an excitable temperament had significantly lower reproductive performance than calmer cows. The modified two‐point chute exit–gait scoring method was repeatable and identified cattle with an excitable temperament.     

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

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.     

Spectral Properties of Near-Earth Asteroids: Evidence for Sources of Ordinary Chondrite Meteorites

Although ordinary chondrite (OC) meteorites dominate observed falls, the identification of near-Earth and main-belt asteroid sources has remained elusive. Telescopic measurements of 35 near-Earth asteroids ( approximately3 kilometers in diameter) revealed six that have visible wavelength spectra similar to laboratory spectra of OC meteorites. Near-Earth asteroids were found to have spectral properties that span the range between the previously separated domains of OC meteorites and the most common (S class) asteroids, suggesting a link. This range of spectral properties could arise through a diversity of mineralogies and regolith particle sizes, as well as through a time-dependent surface weathering process.     

Fine-Scale Doppler Radar Observations of Tornadoes

Observations obtained with a mobile pencil-beam Doppler radar revealed many previously unresolved structures within tornadic storms and tornadoes and helped verify various aspects of conceptual models. Radar data from the parent circulations indicate the existence of spiral reflectivity bands, intense radial wind shear zones, and multiple larger-scale velocity maxima. Tornado structures observed include debris shields, clear axial (eye) regions, multiple reflectivity bands surrounding the center of the eye, and occasional reflectivity protrusions into the eye. Velocity and reflectivity data from tornado-scale circulations show evidence of axial downdrafts.     

Evaluation of tumescent local anesthesia in cats undergoing unilateral mastectomy

ObjectiveTo evaluate the analgesic efficacy and safety of tumescent local anesthesia (TLA) in cats undergoing unilateral mastectomy.Study designProspective clinical trial.AnimalsA total of 12 ovariohysterectomized female cats.MethodsAll animals were premedicated with pethidine (4 mg kg^–1) intramuscularly (IM), followed by induction of anesthesia with propofol (5 mg kg^–1) intravenously and maintenance with isoflurane in oxygen. A refrigerated TLA solution (15 mL kg^–1, 8 °C) was injected using a Klein cannula. The solution was composed of 0.5 mL of epinephrine (1 mg mL^–1) and 40 mL of 2% lidocaine added to 210 mL lactated Ringer’s solution (final lidocaine concentration 0.32%). Heart and respiratory rates, systolic arterial blood pressure, temperature and oxygen saturation were measured during anesthesia. Blood samples were collected from the jugular vein for measurement of plasma lidocaine concentration using high performance liquid chromatography. Postoperative pain scores were evaluated hourly for 6 hours. Analgesic rescue was performed with tramadol (2 mg kg^–1) IM and meloxicam (0.15 mg kg^–1) subcutaneously.ResultsPlasma lidocaine concentration peaked at 90 minutes after injection of TLA, but no concentration considered toxic for the species was measured. The median postoperative analgesia time was 6 hours after injection of TLA.ConclusionsThis study found that TLA prevented sympathetic response to noxious stimuli during anesthesia and provided satisfactory postoperative analgesia in cats submitted to total unilateral mastectomy, with no apparent signs of toxicity.Clinical relevanceTLA can prevent sympathetic stimulation resulting from noxious stimuli during anesthesia, promoting good intraoperative conditions, proving to be a viable addition to analgesia in cats submitted to a total unilateral mastectomy.