Effect of heterologous seminal plasma and semen extenders on motility of frozen-thawed ram spermatozoa

Ram seminal plasma increases the fertility of frozen-thawed ram spermatozoa deposited into the cervix. The aim of the current study was to compare the effect of ram seminal plasma to that of bull seminal plasma, dog prostatic fluid, protein-free TALP TrilEq (Triladyl with 0.5 mt of Equex STM paste added to each 100 mt) and heat-treated skim milk on longevity and percentages of progressively motile and aberrantly motile frozen-thawed ram spermatozoa. Three ejaculates from each of 6 rams were extended in TrilEq, pooled and frozen in straws as a single batch per ram. One hundred and eight straws (3 straws from each ram for each fluid) were thawed in random order. Once thawed, a straw was emptied into a tube with 0.85 ml of the appropriate fluid at 37 degrees C and kept at that temperature for 6 h. Motility was assessed at x200 magnification immediately (time zero) and 2, 4 and 6 h after thawing. Progressive motility decreased from each time to the next (P < 0.05) and was 39.0 % (0 h), 26.0 % (2 h), 19.6 % (4 h) and 12.6 % (6 h); SEM 1.24, n = 108 for each group. Ram seminal plasma resulted in higher progressive motility than bull seminal plasma, lower than milk, and similar to the other fluids. Ram seminal plasma resulted in lower aberrant motility than protein-free TALP and similar aberrant motility to other fluids. The effect of ram seminal plasma and dog prostatic fluid was very similar. The effect of ram seminal plasma on the fertility of frozen-thawed ram spermatozoa deposited into the cervix is not due an exceptionally beneficial effect on the motility of spermatozoa.     

Conception rates in ewes after AI with ram semen preserved in milk-egg yolk extenders supplemented with glycerol

This study aimed at comparing the effect of ram semen preserved at 5°C on two milk‐based extenders (UHT skim milk or INRA‐96^®, 5% egg yolk) supplemented with 2% glycerol, and the preservation time (24 and 48 h) on conception rates after cervical AI of ewes. In two field trials, 1198 Merino ewes were cervical AI in spontaneous oestrus. In Experiment 1, pooled semen (6 rams) was extended in UHT‐base (fresh, control) or chilled for 24 h in UHT5Y (UHT‐base 5% egg yolk), INRA5Y (INRA‐96^® 5% egg yolk), UHT5Y2G (UHT5Y 2% glycerol) or INRA5Y2G (INRA5Y 2% glycerol). In Experiment 2, AI was performed with pooled semen (7 rams) used fresh (extended in UHT‐base or UHT5Y2G, control groups) or chilled (extended in UHT5Y2G) for 24 or 48 h. Conception rate was determined by ultrasound 40 days after AI. INRA‐96^®– had similar conception as UHT‐preserved semen (56.7 vs 55.4%, p > 0.05). Addition of 2% glycerol did not modify the results (56.8 vs 55.2%, p > 0.05). Fresh semen extended in UHT‐base, and UHT5Y2G yielded similar conception rates (60 vs 64%, p > 0.05). Preservation for 24 or 48 h in UHT5Y2G gave similar results (49 vs 47%; p > 0.05). In conclusion, ram semen chilled for 24 h in UHT‐ or INRA‐96^®‐based extenders yielded similar results, and glycerol addition did not have a detrimental effect. UHT5Y2G might be used to extend ram semen for fresh AI, or to preserve it for 24 or 48 h with acceptable results.     

Chilled storage of ram semen improves with the addition of egg yolk and glycerol to milk-based extenders

This study aimed at comparing in vitro, ultra‐heat‐treated (UHT) skim milk and INRA‐96^®‐based extenders supplemented or not with 5% egg yolk and/or 2% glycerol on sperm quality parameters along 72 h of preservation at 5°C, using a factorial design. Semen from six healthy mature Merino rams was pooled and extended in each medium using a split sample procedure (six replicates) and chilled. Subjective motility (SM) (%), membrane integrity (MI) (%) and uncapacitated spermatozoa (US) (×10⁶ spermatozoa/AI dose) were used to assess the semen quality at 0, 12, 24, 48 and 72 h of preservation. UHT‐based extenders yielded better (p < 0.05) SM and MI than INRA‐96^®‐based extenders (59.7% vs 57.9%; 60.2% vs 55.8%, respectively) but similar numbers of US (64.2% vs 62.3 × 10⁶ sperm/AI dose, respectively) along the preservation time. Egg yolk–glycerol or just egg yolk as additives improved (p < 0.05) the results compared with the base extenders without additives or just with glycerol. The sperm parameters assessed decline slowly from 0 to 48 h, with a sharp decline (p < 0.05) at 72 h of preservation. In conclusion, UHT and INRA‐96^® were similar as base extenders, and the addition of 5% egg yolk plus 2% glycerol or just 5% egg yolk improved the quality of ram semen preserved at 5°C, at least for 48 h. The combination of egg yolk–glycerol might provide extra protection in case of fluctuation of temperatures below 5°C, commonly seen under field conditions.     

Influence of idebenone on ram semen quality stored at 4°C

The current study was designed to investigate the effect of idebenone (Id), an antioxidant on ram semen quality. Semen samples were collected, pooled and diluted in a Tris‐based extender supplemented with 0, 1, 2, 4 and 8 µM idebenone. Computer‐assisted sperm analysis was used to evaluate spermatozoa kinematics. Sperm viability and membrane functionality were assessed respectively, by eosin‐nigrosin staining and HOS test. Biochemical assays were carried out to measure different metabolites in spermatozoa and medium at 0, 24, 48 and 72 hr. Total and forward progressive motility were greater in 1, 2 and 4 µM idebenone treated groups compared to control at 24, 48 and 72 hr time points (p < 0.05). Semen supplementation with Id significantly increased viability and functionality of spermatozoa membrane during storage (p < 0.05). Lower amounts of lipid hydroperoxides in medium and spermatozoa were observed in Id‐treated groups compared to control one at 24 and 48 hr of storage (p < 0.05). Medium and spermatozoa amounts of malondialdehyde and nitric oxide were less in Id 4 µM group compared to the control at 72 hr (p < 0.05). Total antioxidant capacity values and superoxide dismutase activity of spermatozoa and medium were greater in 2 and 4 µM idebenone treated groups in comparison with the control at 72 hr (p < 0.05). Results of the current study indicated that ram semen supplementation with Id at 4 µM level improved quality by ameliorating nitrosative and peroxidative stress, hence could be considered as an antioxidant additive during storage at 4°C.     

Influence of pre-insemination douching and different semen extenders on fertility and hatchability of white Leghorn eggs.

1. White Leghorn hens were artificially inseminated twice weekly with undiluted semen or semen diluted (1:3) in Tris buffer, pH 6.8 or saline solution (9 g NaCl/l); with or without pre-insemination douching of the vagina with a solution of ampicillin. 2. Pre-insemination douching reduced the bacterial load of the vagina, resulting in an improvement in fertility and hatchability, with a proportionate reduction in the embryonic mortality. 3. The fertilising and hatching abilities of saline-diluted semen were comparable with those of undiluted semen. 4. Dilution of semen with Tris buffer significantly reduced fertility, but not hatchability.     

Comparison of two different centrifugation extenders for preservation of frozen equine semen

This study compares a commercial semen extender (control group) to ultra high temperature (UHT) skimmed milk (treatment group) used during centrifugation for subsequent cryopreservation of equine semen. Following post‐thawing of semen samples parameters measured included motility, sperm motion kinetics (using computerised assisted semen analysis) as well as acrosome and plasmatic membrane integrity (using fluorescent dyes). After collection and analysis, the sperm‐rich fraction was divided and diluted with either: control (1:1 dilution in a skimmed milk‐glucose extender) or treatment (1:1 dilution in UHT skimmed milk). The milk used in this experiment was of the same source, commercial brand, of only one lot. After dilution, samples were subjected to centrifugation at 600 g for 10 min and sperm pellets were resuspended in a freezing extender to a concentration of 200 × 10⁶ cells/ml. Aliquots were packed into 0.5 ml straws placed in a stainless steel support and kept inside the refrigerator (5°C) for 20 min. Subsequently, these straws were placed at a height of 6 cm over liquid nitrogen for 20 min in an isotherm box. No significant differences were observed in total sperm motility (42.71 vs. 38.29%), progressive sperm motility (12.29 vs. 7.86%), plasma membrane integrity (53.43 vs. 60.14%) or acrosomal membrane integrity (93.29 vs. 93.71%) with a P>0.05 calculated between the control and the treatment groups, respectively. Considering that UHT skimmed milk has a lower cost than the commercial semen extender, this could be an option used during the centrifugation protocol to decrease the expense of the equine semen cryopreservation process and increase shelf life.     

Effect of vaginal douching and different semen extenders on bacterial load and fertility in turkeys

1. A study on artificial insemination of Beltsville Small White turkeys investigated the effect on bacterial load and fertility of vaginal douching with diluents containing Gentamicin 400 microg/ml and different semen extenders. 2. Irrespective of the extenders used, vaginal douching with Gentamicin reduced the microbial load of the vagina with resultant improvement in fertility and hatchability and corresponding reduction in embryonic mortality. 3. Eggs from hens inseminated with semen extended with Beltsville Poultry Semen Extender (BPSE) diluent along with vaginal douching showed a trend towards higher per cent fertility and per cent hatchability of total and fertile eggs set compared to other extenders, though this was non-significant.     

不同稀释剂对梅山猪精液冷冻保存效果的研究

为了探讨稀释剂对梅山猪精液冷冻保存效果的影响,本文选用3种不同的常温稀释液进行常温保存试验,结果发现配方Androhep(A)、Schonow(B)优于葡-柠-EDTA(C)。选用常温配方A、B与3种冷冻稀释液进行配伍组合试验,应用液氮熏蒸法制成试管冻精,结果表明:1A稀释液组合在精子活力和顶体完整率均优于其他组合,解冻后精子活率最高达0.55;不同时间点保存效果检测结果显示,解冻后精子活力和顶体完整率指标稳定。     

犬精液液态保存稀释液及温度筛选试验

探讨了8种稀释液在5℃、10℃、15℃和20℃下保存犬精液的效果。结果表明:15℃为犬精液液态保存的较适温度,3号液为优等液,其中3号液在15℃精子的存活时间为105±10h,与对照液在15℃的保存效果差异极显著(p<0.01)。2、4、5号液是良等液,在15℃精子的存活时间是64±8h,与对照液在15℃的保存效果差异显著(p<0.05)。中等液为6号液,在15℃、20℃保存时与对照液没有统计学上的差异(p<0.05),在其他2种温度保存时不及对照液。7、8号液为差等液,在15℃保存时与对照液差异不显著(p<0.05),在其他3种温度时不及对照液。     

犬精液液态保存稀释液及温度筛选试验

试验研究了 8种稀释液在 5℃、10℃、15℃、2 0℃下保存犬精液的效果。结果表明 ,15℃为犬精液液态保存的较适温度 ,3号液为优等液 ,其中 3号液在 15℃精子的存活时间为 10 5h± 10h ,与对照液在 15℃的保存效果差异极显著 (P <0 0 1)。 2、4、5号液是良等液 ,在 15℃精子的存活时间是6 4h± 8h ,与对照液在 15℃的保存效果差异显著 (P <0 0 5 )。中等液为 6号液 ,在 15℃、2 0℃保存时与对照液没有统计学上的差异 (P >0 0 5 ) ,在其他 2种温度保存时不及对照液。 7、8号液为差等液 ,在 15℃保存时与对照液差异不显著 (P >0 0 5 ) ,在其他 3种温度时不及对照液     

Cryopreservation of ram semen: Manual versus programmable freezing and different lengths of equilibration

The aim of our study was to examine effects of the length of semen equilibration as well as two freezing techniques on ram sperm post-thaw quality. The ejaculates of Wallachian sheep rams (n = 12) were collected by an electro-ejaculation, equilibrated in a Triladyl® (0, 2, 4, 6, and 8 h) containing glycerol and egg yolk and frozen by programmable freezing (PF) or manual freezing (MF). After thawing, sperm samples were subjected to the motility (computer-assisted sperm analysis [CASA]), viability (SYBR-14/PI), and fertilizing ability (FA) (in vitro penetration/fertilization test on bovine oocytes) assays. It was found that the equilibration of 6 h (E-6) ensured higher post-thaw sperm motility and progressive movement compared with other lengths tested, irrespective of a freezing technique. The E-6 sperm viability did not differ between PF and MF but was lower (P < 0.05) than control. Sperm FA (E-6) was similar in PF (60.44%) and MF (62%) but slightly lower than in fresh (72.8%). Our data demonstrate that the use of MF was comparable with PF, which can be applied in the field conditions without need in a piece of cost-expensive equipment, which can greatly benefit the gene bank of animal genetic resources.     

保持公羊精液卫生无菌的措施

公羊包皮、四肢被毛、母台羊、采精及精液处理环境条件、饲养条件、人员操作、精液稀释液等十大因素可能是造成精液污染的主要因素。因此,必须从上述各方面采取技术措施保持精液卫生无菌。     

Changes in proacrosin levels during preservation of ram semen

The percentual change in the content of pro-acrosin taking place in ram semen preserved for a short and long time was examined in the period from April to October. Two diluents for keeping semen at the temperature of 16 degrees C and one diluent for keeping semen at 3 to 4 degrees C were used in short-time preservation. The content of pro-acrosin was measured 2, 8 and 12 hours after dilution. The lactoso-yolk diluent and the diluent after Milovanov (1980) were used for cryopreservation. The content of pro-acrosin was examined before and after semen freezing. In short-time preservation, no statistically significant decrease of pro-acrosin content was demonstrated in the H Milch diluent (Peter, 1975) at the storage temperature of 16 degrees C and in the diluent after Milovanov (1980) at the temperature of 3 to 4 degrees C. In the diluent prepared after Milovanov (1980) a significant decrease of pro-acrosin content during preservation was recorded at the storage temperature of 16 degrees C. When the short-time preservation diluents were compared, significant differences in pro-acrosin content were found between them. In the long-time preservation diluents a significant difference in pro-acrosin content was found before and after semen freezing; the difference between the short- and long-time preservation diluents was also significant. A positive correlation was found between sperm activity and pro-acrosin content.