Lake Extender Research Articles

Supplementation of Rooster Semen Extender with Aqueous Extract of Urtica dioicafor a Long Time Preservation by Low Temperature.

The peroxidation of spermatozoa membrane phospholipids is a primary cause of irreversible changes in the preservation of avian semen. To address this issue, the objective of the present study was to assess the potential of Urtica dioica extracts in protecting avian spermatozoa during prolonged storage. Gas chromatography-mass spectroscopic techniques were employed to evaluate the bioactive compounds present in the aqueous and ethanolic extracts obtained from the aerial parts and roots of U. dioica. Semen samples were collected from 16 roosters twice a week and were diluted in Lake's extender containing different concentrations (0, 0.5, and 1 mg/100 mL) of the various extracts. Subsequently, the extended semen samples were cooled and stored at 5°C, and the sperm quality parameters were assessed at 0, 12, 24, and 36 hours of storage. The data from this experiment clearly demonstrate that the addition of nettle root aqueous extracts to the semen diluent, especially at a concentration of 0.5 mg/100 mL, resulted in a significant improvement in various sperm quality parameters. Notably, there were enhancements in total and progressive sperm motilities, viability, fertility, membrane integrity, acrosomal membrane integrity, and a reduction in malondialdehyde production in rooster semen stored in vitro for up to 36 hours. Interestingly, the present study reveals that the beneficial effects of the aqueous extracts from different parts of the nettle were supported not only by the conventional manual method but also by the computer-assisted sperm analysis system. This dual confirmation further emphasizes the positive impact of the aqueous extract on various sperm traits during cooled semen preservation. In conclusion, this study highlights the potential of U. dioica extracts, particularly the aqueous extract from nettle roots at a concentration of 0.5 mg/100 mL, in safeguarding avian spermatozoa during prolonged storage. The significant improvements in various sperm quality parameters and the validation of results through both manual and computer-assisted analysis methods provide strong evidence for the application of U. dioica extracts in avian breeding programs and artificial insemination practices.

Type III antifreeze protein (AFP) improves the post-thaw quality and in vivo fertility of rooster spermatozoa

Antifreeze proteins (AFP) have the potential for improving sperm cryopreservation. We have applied Type III antifreeze protein (AFP3) on the cryopreservation of spermatozoa from broiler breeder roosters, aiming to enhance post-thawing quality and fertility. Semen was extended at 37°C in Lake's extender containing AFP3 at 0.01, 0.1, 1, 5, and 10 µg/mL (no AFP3 as control). Post-thawing sperm assessment included sperm motility (CASA), morphology, membrane functionality by hypoosmotic swelling test (HOST), lipoperoxidation as malondialdehyde (MDA) production, and sperm viability, early apoptosis (phosphatidylserine exposure as annexin V-positive staining in viable spermatozoa), and mitochondrial activity by flow cytometry. Fertility was assessed after artificial insemination (30 hens/treatment). Total and progressive motility, membrane functionality, and mitochondrial activity increased in 0.1 and 1 µg/mL AFP, compared to control and other concentrations, whereas apoptosis was significantly lower. VAP, VSL, and viability were significantly higher for 1 µg/mL AFP3 than with the other treatments except for 0.1 µg/mL (which was not always significantly different from the control or other concentrations), and with abnormal forms being significantly lower. The proportion of fertilized and hatched eggs was also higher for 1 µg/mL AFP3, with 0.1 µg/mL also showing significantly higher results than the control, and no differences with other concentrations). In conclusion, 1 µg/mL AFP3 could improve the post-thawing results of rooster spermatozoa frozen in Lake's extender. According to our results, concentrations between 1 and 0.1 µg/mL could be similarly efficient.

117 CRYOPRESERVATION OF SNAKE SEMEN: ARE WE FROZEN IN TIME?

The increasing number of endangered snake species, isolation of small fragmented populations with associated inbreeding and mating or conception problems in captivity underscore the need to develop assisted reproductive techniques such as semen cryopreservation and artificial insemination to enhance conservation efforts. However, no efficient protocols for semen evaluation, cooling, or freezing are described in the 4 known publications on snake semen cryopreservation. In this initial study, semen was collected noninvasively from 4 live adult red diamond rattlesnakes (Crotalus ruber) by ventral massage. An aliquot of each ejaculate was diluted in Ham's F10 and evaluated for motility and speed of progression (SOP, scale of 1–5, 5 being fastest) before and after freezing. Because ejaculate volumes were very small, each one was subjected to 2 to 4 treatments only. Samples were diluted in Lake's extender with 2 or 4% dimethyl sulfoxide or Test-Yolk extender with 4%, 8%, or 10% glycerol before cooling to 4°C. Semen extended with Lake's (a common avian semen extender) was then frozen by one of two protocols (FP): FP1, pelleted in liquid nitrogen vapor; FP2, placed in cryovials in an alcohol bath for 4 h in a –80°C freezer (cooling rate of 1°C min–1). Semen extended with Test-Yolk (a widely used mammalian semen extender) was frozen by one of two protocols: FP3, placed in cryovials in a controlled-rate freezer and cooled at 1°C min–1 to –20°C, then 50°C min–1 to –80°C; FP4, placed in cryovials in a controlled-rate freezer and cooled at 1°C min–1 to –8°C, then 3°C min–1 to –16°C, then 5°C min–1 to –80°C. Interestingly, semen frozen in Lake's extender exhibited no post-thaw motility, regardless of dimethyl sulfoxide concentration or FP (Table 1). In contrast, all semen frozen in Test-Yolk exhibited motility post-thaw. Semen from male 42 frozen by FP3 with 8% glycerol resulted in the greatest post-thaw motility of all treatments for both males. However, the same treatment for male 122 was not as successful, suggesting a possible male effect on semen cryosurvival (Table 1). The single sample frozen with 4% glycerol exhibited the lowest post-thaw motility, indicating that higher concentrations of the cryoprotectant may be beneficial to snake sperm. The exciting results of this preliminary study may guide future research in the long-neglected field of snake semen cryopreservation. Table 1.Motility/speed of progression for fresh and cryopreserved semen of red diamond rattlesnakes frozen with Test-Yolk extender and glycerol

The Effect of Semen Dilution on Morphology and Fertility of Japanese Quail (<I>Coturnix coturnix japonica</I>) Spermatozoa

The effect of quail semen dilution with three extenders : Lake's, Ringer's and Tyrod's on morphology and fertility trials of unstored spermatozoa was evaluated. Semen was collected twice a week by male stimulation by female method, from 60 quail males randomly divided into four groups (I-IV). Forty-eight females divided into four respective groups (A-D) were inseminated intravaginally with 10μl of fresh or 20μl of 2-folds diluted semen. Semen dilution did not affect the number of live spermatozoa in total, but the number of live, morphologically normal spermatozoa depended on the diluent. The highest number of morphologically normal spermatozoa -65.3% vs. 67.5% in fresh semen was stated in semen diluted with Lake's extender. It was significantly higher (P≤0.01) than in semen diluted with Ringer's diluent (56.2%). Egg fertility (99.2%), hatchability from set (91.2%) and fertile eggs (93.9%) in naturally mated group were significantly higher (P≤0.01) when comparing with females inseminated artificially both, with fresh and diluted semen. The highest fertility (60.1%) was obtained for spermatozoa diluted with Lake's extender, the lowest for spermatozoa treated with Ringer's solution (30.2%).

The effect of DMA level on morphology and fertilising ability of Japanese quail ( Coturnix japonica) spermatozoa

The effect of different levels (2, 4 or 6%) of DMA (dimethylacetamide) on the morphology and fertilising ability of unfrozen quail spermatozoa was evaluated. Semen was collected from 72 males kept individually in cages and randomly divided into four groups: Group I—control – fresh undiluted semen (12 males) and three experimental groups (20 males each) – semen diluted 1:1 with Lake's extender and supplemented with 2% (Group II), 4% (Group III) or 6% (Group IV) of DMA (final concentration). Sperm morphology was evaluated at each step of semen preparation, i.e. in fresh and diluted semen, semen supplemented with DMA and semen that remained after insemination. For fertility tests, 36 females were divided into four groups (nine females each). Females in the control group were inseminated with 10 μl of fresh semen, in the experimental groups with 40 μl of diluted semen. Each stage of quail semen treatment had a deleterious effect on sperm morphology. The highest percentage of morphologically normal cells in semen evaluated after insemination, was observed in samples with 2% DMA, and the lowest—in samples with 6% DMA. Semen dilution and DMA addition significantly affected the fertilising potency of spermatozoa. Fertility of eggs collected from the control group (71.5% on average) was significantly higher ( P ≤ 0.01) compared to experimental groups (Group II: 2.3%; Group III: 3.9%; Group IV: 2.8%). However, differences between the experimental groups were not significant ( P ≤ 0.05).