Cryopreservation of Ram Cauda Epididymal Spermatozoa Using Different Buffers and Sugar Combinations

The aim of this experiment was to study the effect of different sugars and buffers combinations in the extenders viz. Tris citric acid fructose (TCF), Tris citric acid glucose (TCG), Sodium citrate fructose (SCF) and Sodium citrate glucose (SCG) on the quality of Cauda epididymal spermatozoa of ram during cryopreservation and post thaw. Spermatozoa were recovered from Cauda epididymidis by incision method. Samples showing ≥70 % progressive sperm motility were pooled. Each pooled cauda epididymal sperm sample was divided into four aliquots and spermatozoa in each aliquot were washed using isotonic buffer by double centrifugation. Washed spermatozoa in each aliquot were extended separately in the four different extenders using 20% egg yolk and 8% glycerol as cryoprotectant. The quality of spermatozoa was evaluated immediately after extension in the particular extenders (pre-freeze) and at post thaw. The percent sperm motility was significantly (p<0.05) higher for TCF (45.00±4.47) than TCG (27.50±6.55) and SCG (20.83±5.39) extenders at post thaw. The percentage of HOST reacted spermatozoa was significantly higher (P<0.05) for TCF (61.05±2.60) than SCF (45.81±4.90) and SCG (46.41±4.16) at post thaw. The percent intact acrosome was also significantly higher (P<0.05) in TCF (79.39±2.16), SCF (80.74±1.38) and SCG (78.34±2.94) than TCG (71.32±2.47) at post thaw. In conclusion, the use of fructose as energy source in the Tris extender (TCF) was found the best combination of buffer and sugar for maintaining higher sperm quality during cryopreservation of ram caudaepididymal spermatozoa

Recovery of sperm after epididymal refrigeration from domestic cats using ACP-117c and Tris extenders / Recuperação de espermatozoides após refrigeração do epidídimo de gatos domésticos utilizando os diluidores ACP-117c e Tris

We aimed to compare fresh sperm and sperm cooled to 4ºC that had been recovered from the epididymides of cats using powdered coconut water (ACP-117c) and Tris extenders. Sixty epididymides were divided into 6 groups: 10 fresh epididymides were recovered using Tris (T0h); 10 were kept at 4°C/2h and recovered using Tris (T2h); 10 were kept at 4°C/4h and recovered using Tris (T4h); 10 fresh were recovered using ACP-117c (A0h); 10 were kept at 4°C/2h and recovered using ACP-117c (A2h), and 10 were kept at 4°C/4h and recovered using ACP-117c (A4h). The testis-epididymis complexes (TEC) control were not cooled. The others were cooled at 4°C for 2 or 4h. The epididymis was separated and the sperm was recovered by the modified flotation method. Sperm kinetic parameters were evaluated by a computer-system analysis, and vigor, viability, concentration, membrane function and morphology of the sperm were assessed under a light microscope. The progressive motility with ACP-117c declined after 2h of cooling, but did not differ between fresh and 4h. The vigor and membrane function were higher in A4h than A0h. The vigor at T2h and T4h were decreased compared to T0h. T0h was higher than A0h for vigor and sperm membrane function. However, after 4h of cooling, ACP-117c maintained a higher percentage of living cells. Feline epididymal sperm quality can be maintained to the degree necessary for artificial breeding programs following cooling and ACP-117c may be successfully used to recover cat sperm that have been cooled for up to 4h.(AU)

Objetivou-se comparar a qualidade de espermatozoides recuperados a fresco e após refrigeração a 4ºC do epidídimo de gatos domésticos utilizando-se os diluidores ACP-117c e Tris. Sessenta epidídimos foram distribuídos em seis grupos: 10 epidídimos a fresco com o Tris (T0h), 10 a 4°C/2h e recuperados com Tris (T2h), 10 a 4°C/4h e recuperados com Tris (T4h), 10 epidídimos a fresco com o ACP-117c (A0h), 10 a 4 °C/2h e recuperados com ACP-117c (A2h), 10 a 4°C/4h e recuperados com ACP-117c (A4h). Os complexos testículo-epidídimo (CTE) do controle não foram refrigerados. Os outros foram refrigerados a 4°C durante duas e quatro horas. Os epidídimos foram separados das demais estruturas, e os espermatozoides recuperados pela técnica de flutuação modificada. Os parâmetros cinéticos foram avaliados em um sistema computadorizado, e o vigor, a viabilidade, a concentração, a funcionalidade de membrana e a morfologia celular foram avaliados em microscopia de luz. A motilidade progressiva com ACP-117c declinou após duas horas de refrigeração, mas não diferiu entre a recuperação a fresco e após refrigeração por quatro horas. Vigor e integridade funcional da membrana celular foram significativamente superiores no grupo A4h em comparação ao A0h. O vigor espermático em T2h e T4h reduziu significativamente em comparação com T0h. T0h foi significativamente superior ao A0h quanto aos parâmetros de vigor e integridade funcional da membrana espermática, entretanto, após quatro horas de refrigeração, o ACP-117c apresentou um maior percentual de células vivas. Os espermatozoides epididimários de felinos domésticos conseguem manter a qualidade necessária para serem utilizados em programas de reprodução artificial após serem refrigerados e recuperados por meio da técnica de flutuação modificada, e o diluidor ACP-117c pode ser utilizado com sucesso para recuperação de células espermáticas refrigeradas de gatos por até quatro horas.(AU)

Cooling of ejaculated and epididymal stallion sperm / Refrigeração de espermatozoides do ejaculado e epidídimo em garanhões

After a serious injury or sudden death, epididymis cauda sperm recovery and cryopreservation may present as the last opportunity to obtain genetic material from a valuable stallion. This study evaluated the viability of cooled equine sperm collected by three different methods: sperm of ejaculated (G1), sperm recovered from the epididymal cauda immediately after orchiectomy (G2) and sperm recovered from the epididymal cauda after storage for 24 hours at 5°C (G3). To obtain G1 sperm, two ejaculates were collected. After 1 week, all stallions underwent a bilateral orchiectomy, and one of the removed epididymides was flushed to obtain G2 sperm. The contralateral epididymis was stored at 5°C for 24 hours before being flushed to obtain G3 sperm. The sperm samples were evaluated immediately after the addition of the refrigeration extender, and after 24 and 48 hours of storage at 5°C. After 24 and 48 hours of storage, the epididymal sperm demonstrated higher motility traits when compared to the ejaculated sperm (P<0.05). These results indicate that sperm recovered from the epididymal cauda of stallions are more resistant to the cooling process, with higher kinetic parameters and plasma membrane integrity when compared to the ejaculated sperm.

A recuperação de espermatozoides da cauda do epidídimo pode ser a última chance para preservação do germoplasma quando ocorre morte súbita ou lesão grave em garanhões de alto valor genético. O presente trabalho comparou a viabilidade após refrigeração dos espermatozoides do ejaculado (G1), recuperados da cauda do epidídimo imediatamente após a orquiectomia (G2) e recuperados após armazenamento do epidídimo por 24 horas a 5ºC (G3). No G1 foram colhidos dois ejaculados. Uma semana após a colheita dos ejaculados os garanhões foram submetidos à orquiectomia bilateral e realizada a colheita dos espermatozoides da cauda do epidídimo de um testículo de cada garanhão (G2). O testículo contralateral permaneceu a 5°C por 24 horas, antes da recuperação espermática (G3). A análise das amostras foi realizada imediatamente após a adição do meio de refrigeração, e após 24 e 48 horas de armazenamento a 5°C. Após 24 e 48 horas de armazenamento, os espermatozoides do epidídimo demonstraram características de cinética maiores que os do ejaculado (P<0.05). Estes resultados indicam que espermatozoides recuperados da cauda do epidídimo foram mais resistentes ao processo de refrigeração, com maiores parâmetros de cinética espermática e integridade da membrana plasmática quando comparados aos espermatozoides do ejaculado.

In-vitro effect of nimbolide, an isoprenoid of neem leaf, on antioxidant system of rat cauda epididymal spermatozoa: A dose dependent study.

The present in vitro study aims to address the possible evaluation of nimbolide, a major component of neem leaves, on the antioxidant system of rat spermatozoa to promote oxidative stress in a dose-dependent manner. To assess the effect of the nimbolide on activities of superoxide dismutase (SOD), catalase, Glutathione reductase (GR), Glutathione peroxidase (GPx), α-Glucosidase, production of Hydrogen peroxide (H2O2) and level of lipid peroxidation (LPO) in rat spermatozoa, increasing quantities 0.5 mM, 1 mM, 1.5 mM and 2.0 mM per ml of the nimbolide was added to the cultured medium prior to the addition of cauda epididymal spermatozoa. The spermatozoa were observed at 6th h post-culture and the expressions of enzyme activities and production of H2O2, level of LPO were recorded. The activities of antioxidant enzymes decreased significantly while the levels of H2O2 generation and LPO increased significantly in nimbolide treated spermatozoa in a dose dependent manner when expressed in terms of milligram protein and milligram DNA. The activity of α- glucosidase, a negative control against antioxidant enzymes, did not show any significant change at any of the doses. The results suggest that graded doses of nimbolide elicit depletion of antioxidant defense system in sperm, indicating nimbolide-induced oxidative stress in the epididymal sperm of rats.