Effect of Egg Yolk and Soybean Lecithin on Tris-Based Extender in Post-Thaw Ram Semen Quality and in vitro Fertility

Abstract

The aim of the current study was to evaluate the effect of egg yolk and different soybean lecithin concentrations on the efficiency of ram semen cryopreservation and to test the fertilizing ability of frozen-thawed ram semen. Ejaculates with a thick consistency, rapid wave motion (3-5), and >70% initial motility were pooled. Pooled semen were then divided into four groups and diluted at 1/5 (semen/extender) with 1%, 3%, 6% lecithin (L1, L3 and L6) or 20% egg yolk (EY20) using the two-step dilution method. As expected, the results of the current study showed that both motility and the rates of defective acrosomes in sperm were negatively affected by the cryopreservation procedure (P<0.001). The motility values of at 5°C and post-thawed semen in the EY20 group were significantly higher than those in the L1, L3 and L6 groups (P<0.05). There were no differences in motility rates among the lecithin groups at the dilution, cooling, equilibration or post thawing stages (P>0.05). The results of in vitro fertilization, as assessed by the rate of blastocyst formation, were more successful in the EY20 group than those noted in different lecithin groups. In conclusion, freezing ram semen with an extender containing egg yolk could yield better post-thaw sperm parameters and embryonic development compared to lecithin containing extenders. Keywords: Cryopreservation, Lecithin, Egg yolk, Ram semen, IVF Tris-Bazlı Sulandırıcılarda Yumurta Sarısı ve Soya Lesitininin Eritme Sonrası Koç Spermasının Kalitesi ve in vitro Fertilite Üzerine Etkisi ÖzetBu çalışmanın amacı, yumurta sarısı ve farklı lesitin konsantrasyonlarının koç spermasının dondurulabilirliği üzerine etkisini değerlendirmek ve dondurup çözdürülen koç spermasının fertilizasyon yeteneğini tespit etmektir. Kitle hareketi (3-5) ve >%70 motiliteye sahip ejakülatlar birleştirildi (pooling). Pooling yapılan sperma dört gruba bölündü ve %1, %3, %6 (L1, L3 ve L6) lesitin veya %20 yumurta sarısı (EY20) içeren sulandırıcılar ile 1/5 (sperma/sulandırıcı) oranında iki aşamalı sulandırma yöntemi kullanılarak sulandırıldı. Bu çalışmanın sonucunda; motilite ve akrozomal bozukluk oranlarının dondurma prosedüründen olumsuz yönde etkilendiği tespit edildi (P<0.001). EY20 grubunun 5°C'de ve eritme sonrası motilite değerleri L1, L3 ve L6 gruplarına göre yüksek bulundu (P<0.05). Sulandırma, soğutma, ekilibrasyon ve eritme sonrası aşamalarda motilite oranları bakımından lesitin grupları arasında farklılık saptanmadı (P>0.05). Blastosist oranları bakımından değerlendirilen in vitro fertilizasyon sonuçlarına göre, EY20 grubunun farklı lesitin gruplarından daha başarılı olduğu tespit edildi. Sonuç olarak, yumurta sarısı içeren sulandırıcı ile dondurulan koç spermasının lesitin gruplarına göre eritme sonrası spermatolojik özellikler ve embriyonik gelişim bakımından daha üstün olduğu tespit edildi.

Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C

“…Providing protection against the cold shock, egg yolk is one of the most common ingredients of semen extenders (29,38). The non-dialyzable protein fractions and low density lipoproteins (lecithin) of egg yolk provide the best protection to ram semen during liquid storage (38).…”

“…The non-dialyzable protein fractions and low density lipoproteins (lecithin) of egg yolk provide the best protection to ram semen during liquid storage (38). Egg yolk contributes to protection from the detrimental effects of cold shock through maintenance of the motility, plasma membrane and acrosome integrity during cryopreservation (38) and liquid storage (16) of ram spermatozoa.…”

“…The non-dialyzable protein fractions and low density lipoproteins (lecithin) of egg yolk provide the best protection to ram semen during liquid storage (38). Egg yolk contributes to protection from the detrimental effects of cold shock through maintenance of the motility, plasma membrane and acrosome integrity during cryopreservation (38) and liquid storage (16) of ram spermatozoa. On the other hand, main disadvantages of using an animal-derived material in semen extender can be listed as: failure to prevent individual differences (lack of homogeneity), risk of contamination and transmission of diseases (16,38).…”

“…Egg yolk contributes to protection from the detrimental effects of cold shock through maintenance of the motility, plasma membrane and acrosome integrity during cryopreservation (38) and liquid storage (16) of ram spermatozoa. On the other hand, main disadvantages of using an animal-derived material in semen extender can be listed as: failure to prevent individual differences (lack of homogeneity), risk of contamination and transmission of diseases (16,38). The low-density lipoprotein fraction such as lecithin (35,38) or multifunctional macromolecules such as bovine serum albumin (BSA) (19,31,33) could be substitutes for egg yolk in a semen extender.…”

“…Although lecithin and BSA supplementation was used as an alternative to egg yolk through improving the preservation conditions of semen extenders (35,38), there are no researches about the efficiency of lecithin-BSA combination for liquid storage of ram semen. The objective of this study was to find out the optimum dose of BSA in lecithin based extenders for improving the motility, viability and acrosome integrity of ram spermatozoa at 5 °C during storage for 72 h.…”