36h睡眠剥夺对雄性小鼠性行为及精子质量的影响

[目的]研究睡眠剥夺对雄性小鼠性行为及精子活性的影响。[方法]用轻柔刺激法睡眠剥夺昆明雄性小鼠36 h,观察其性行为,通过镜检计算精子活率和精子畸变率。[结果]36 h睡眠剥夺后,捕捉潜伏期极显著升高(P0.01),捕捉次数极显著降低(P0.01)。36 h睡眠剥夺后精子活率显著降低(P0.05),精子畸变率极显著升高(P0.01)。[结论]36 h睡眠剥夺可对雄性小鼠性行为及精子质量产生明显不良影响。     

The Use of Sex-Sorted Stallion Semen in Embryo Transfer Programs

Sorting stallion semen into two separate populations of enriched X- or Y-bearing sperm can be done successfully. For this, stallion semen can be shipped to a sorting facility, but the mare must be in close to the sorting laboratory. Fertility rates when using 20-40 million sperm are an acceptable 60% per insemination. The procedure can be implemented in embryo transfer programs, with no deleterious effect on the pregnancy rate or embryonic death.     

Hyperactivated Sperm Motility: Are Equine Sperm Different?

A recent study has suggested a central role for hyperactivated sperm motility in successful equine in vitro fertilization, as the highest apparent fertilization rates reported yet were obtained using exogenous stimulation of hyperactivated motility in sperm that had been incubated for capacitation. Hyperactivated motility has been studied intensively in other species, but little data are available in this area in the horse. Hyperactivated motility is associated with an influx of calcium from the extracellular space, and in other species this occurs because of an increase in intracellular pH during capacitation. Influx of calcium seems to trigger changes in flagellar function through calcium–calmodulin–calmodulin kinase-related effects on dynein function, but the exact mechanisms resulting in hyperactivated motility are still unclear. We have been exploring the physiology of hyperactivated motility in stallion spermatozoa and have found many of the mechanisms present in other species to be in place in the stallion. Further research is needed to determine why stallion spermatozoa seem to fail to undergo hyperactivation in response to capacitating environments that support this activity in other species.     

Measurement of Concentration and Viability in Stallion Sperm

Sperm concentration and sperm membrane intactness (SMI) or viability are two measures of sperm quality that provide important but different information about a stallion's reproductive capability. Sperm concentration is a measure that, by itself, informs little about the reproductive status of either the stallion or the ejaculate. Nevertheless, it is part of the product, along with semen volume, that determines total sperm number. The correct calculation of total sperm number directly affects the number of mares a stallion can breed and therefore, fertility. If either sperm concentration or semen volume is incorrectly measured, both the number of mares that a stallion can breed and the fertility of those breedings are affected. Although considerable between-stallion variation exists, sperm concentration, semen volume and total sperm number tend to be seasonal and vary with ejaculation frequency.     

Effects of Pentoxifylline on Equine Epididymal Sperm

This study evaluated whether pentoxifylline (PTX) present in the flushing extender influenced the function of equine epididymal spermatozoa after recovery and after thawing. For this experiment, 58 testicles from 29 Brazilian Jumping Horses were used. Cauda epididymides of each stallion were separated and flushed with a skim milk extender, with or without 7.18 mM PTX and then subjected to the freezing process. Samples flushed with the extender containing PTX showed a significant increase in total motility, progressive motility, straight line velocity, curvilinear velocity, and percentage of rapid sperm immediately after the recovery of epididymal sperm and after 15 minutes of incubation at 37°C (P < .05). However, the presence of PTX in the flushing extender did not affect the post-thaw motility parameters or plasma membrane integrity (P > .05). The results of this study showed that the PTX present in the flushing extender improved motility parameters of recently recovered epididymal sperm and had no deleterious effects on plasma membrane integrity and freezability of equine epididymal sperm.     

The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen

The aim of this study was to determine the synergistic effects of centrifuged egg yolk (EY) and soybean lecithin on post-thaw Caspian horse sperm motility, morphological abnormalities, and assessment of membrane integrity. The centrifuged EY (CEY) was added at concentrations of 2% and 4% to a defined INRA plus 1.25% soybean lecithin extender used to freeze Caspian horse semen. In this experiment, ejaculates collected from each Caspian horse (n = 4) were divided into three equal aliquots and diluted in CEY 2% (INRA2), 4% (INRA4) supplemented, and without any CEY (INRA0) in INRA plus 1.25% soybean lecithin extender, respectively. Thereafter, samples were frozen and thawed following a standard protocol. Sperm cryosurvival was evaluated in vitro by microscopy assessments of post-thaw sperm motility (by means of computer-assisted semen motility analysis [CASA]), acrosomal and other abnormalities (head, mid-pieces, and tail) and plasma membrane integrity (evaluated by HOST). In Caspian stallion, semen extended with INRA2 had significantly higher CASA motility and CASA progressive motility than those extended with the rest of extenders after freezing and thawing (P < .001). There was no significant difference in path velocity (VAP), VCL, and ALH among three groups (P > .05). For straight line velocity (P < .01) and LIN (P < .001), the highest values were obtained from the INRA4 group. The highest percentages of acrosomal and other abnormalities were found in semen diluted in INRA4 (P < .001). In the group frozen INRA2, the percentage of membrane integrity was significantly higher than that of the other groups (P < .001). The use of CEY 2% in combination with soybean lecithin significantly improved Caspian horse semen freezability.     

The Effects of an Oxygen Scavenger and Coconut Water on Equine Sperm Cryopreservation

Alternative sources of lipoproteins in semen extenders could replace animal by-products. We hypothesized that: (1) post-thaw semen parameters and fertility would not be different in coconut water (CW)–treated samples compared with egg yolk (EY)–treated samples and (2) the use of an oxygen scavenger (Oxyrase) would improve post-thaw sperm motility and membrane integrity and decrease lipid peroxidation. Experiment 1: three ejaculates each from five stallions were split into four treatments: EY, CW, egg yolk with Oxyrase, and coconut water with Oxyrase. Computer-assisted sperm analysis measured progressive and total motility, velocity, and linearity. Membrane integrity, apoptosis, and lipid peroxidation were evaluated using propidium iodide, annexin, and BODIPY fluorescent probes, respectively. Samples were cryopreserved, stored in liquid nitrogen, and then thawed to 37°C and analyzed again. Experiment 2: one ejaculate was divided into two aliquots and cryopreserved using either CW or EY. In a crossover design, 12 mares were bred on two consecutive cycles with either EY or CW. Pregnancy evaluations were at 14-day gestation. No differences were detected in sperm parameters between CW and EY (P > .05). Oxyrase did not improve sperm motility parameters in post-thaw samples, nor did it show protective effects for viability or against membrane damage (P > .05). More mares became pregnant using CW than EY (11/12 vs. 6/12, respectively; P = .013). Use of CW is a viable alternative to animal-based products in the cryopreservation of stallion semen.     

High or Low Body Fat Deposition in the Presence of a Normal Oral Sugar Test is Not Associated With Postthaw Semen Parameters in Stallions

This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallions. One ejaculate from each stallion was frozen with a commercial egg yolk-based extender. Postthaw sperm motility parameters, plasma membrane integrity, mitochondrial membrane potential, hydrogen peroxide and intracellular superoxide production, and lipid peroxidation were analyzed for all stallions. The circumference at 25% and 50% of the neck’s length were larger for High-BCS stallions (P < .05). There were no differences between groups for the neck crest height (P > .05). Stallions with High BCS had greater SFT at the tail head than stallions with Low BCS (P < .05); however, there were no differences between groups in the SFT at the shoulders and withers (P > .05). All stallions had resting blood glucose below the cutoff for equine metabolic syndrome. There were no differences between groups for resting glucose concentrations or for a peak at 30 or 60 minutes after initiation of the OST (P > .05). There were no differences in sperm parameters between groups (P > .05). Collectively, the findings of the present study suggest that High BCS or Low BCS in the presence of normal OST do not explain post-thaw semen parameters.     

Stallion Semen Incubated with Hydrogen Peroxide Decreased DNA Fragmentation as Measured by the TUNEL Assay

Spermatozoa are highly specialized cells that are sensitive to environmental factors such as temperature and redox state. Hydrogen peroxide (H₂O₂) is a reactive oxygen species, and its presence is often associated with a decline in sperm function. The aim of this study was to evaluate the effect of H₂O₂ and heat stress on DNA damage, membrane integrity, and motility of stallion spermatozoa. Spermatozoa from three light-horse stallions were subjected to thermal stress, treatment with H₂O₂, or a combination of both. Treatments were organized using a 2 × 2 factorial design consisting of heat stress (control, 41°C) and potential oxidative stress (control, 50 μM H₂O₂) for 1 hour. The experiments were repeated independently on all stallions. Primary motility parameters were measured using a computer-assisted sperm analysis, whereas DNA damage was assessed using the TUNEL assay. To evaluate membrane integrity, an amine reactive dye was utilized. DNA and membrane integrity were simultaneously assessed in the same flow cytometry assay. Sperm incubated at 41°C were observed to have decreased motility (76.2% vs. 66.6%; P < .05) but not progressive motility (39.2% vs. 25.6%), membrane damaged (30.8% vs. 37.4%), or DNA damage (19.7% vs. 17.3%). Interestingly, when compared to control, treatments with H₂O₂ had decreased DNA damage (19.7% vs. 7.1%; P < .05), but did not affect any other parameter. Although our experiment demonstrated a favorable effect of 50 μM H₂O₂ on DNA damage, further studies need to be conducted to confirm these findings and to clarify any possible signaling mechanisms.     

Diagnostic Methods for Evaluation of Stallion Subfertility: A Review

Classically, evaluation of the breeding stallion for reduced fertility has relied on physical examination of the reproductive system, as well as evaluation of sperm number, motility, and morphology. Over the past 20 years, a number of other diagnostic methods have become available to facilitate reproductive evaluation of the stallion. Specifically, ultrasound imaging has provided much-improved diagnostic methods for evaluation of the external and internal genitalia of the stallion, and these methods have now become routine in evaluation of the stallion. Biochemical analyses of semen can provide useful information for diagnosis of azoospermia (determination of alkaline phosphatase), detection of urine contamination, or changes in pH. Numerous sperm function assays provide information concerning subcellular compartments of the sperm including the plasma membrane, DNA, acrosome, and mitochondria. Data correlating these functional assays with fertility in the stallion are limited in most cases, with the exception of the sperm chromatin structure assay. Finally, the recent sequencing of the equine genome offers the possibility of both marker-assisted selection for fertility traits and more specific information about genetic mutations that may be associated with differing levels of fertility in the stallion.