Effect of Warming Regimen on Canine Epididymal Spermatozoa Following Vitrification Using a Coconut Water Extender Supplemented with Egg Yolk

Technical Summary: Effect of Warming Regimen on Canine Epididymal Spermatozoa Following Vitrification

Problem Statement
The preservation of canine genetic material via epididymal sperm recovery and vitrification is increasingly vital for breeding programs and germplasm conservation, particularly for animals undergoing orchiectomy, unexpected death, or endangered species management. While conventional slow freezing remains the standard for canine semen, it often yields variable post-thaw survival. Vitrification, an ultra-rapid solidification technique, offers a promising alternative but requires precise protocol optimization. A critical, yet under-investigated, factor in vitrification success is the warming (devitrification) procedure. While previous studies have explored warming rates for ejaculated canine semen, the specific influence of different slow-warming temperatures on epididymal spermatozoa—which differ biologically from ejaculated sperm in membrane composition and lack seminal plasma—remains unclear. Furthermore, the efficacy of natural extenders like coconut water, combined with egg yolk, specifically for vitrifying epididymal sperm has not been fully established.

Methodology
The study utilized epididymal spermatozoa collected from twelve clinically healthy dogs of various breeds undergoing routine orchiectomy at Trakia University. Sperm were recovered using a float-up method from the cauda epididymis and ductus deferens.

• Extender Formulation: Samples were processed in a coconut water-based extender (50% coconut water, 25% distilled water, 25% 5% anhydrous monosodium citrate) supplemented with 20% egg yolk. Coconut water was sourced from certified organic green coconuts.
• Vitrification: After equilibration at 4°C for 60 minutes, 33 µL aliquots were vitrified by direct dropping onto a stainless-steel strainer above liquid nitrogen (LN₂). Samples were stored in LN₂ for 7 days.
• Warming Regimens: Following storage, samples were devitrified using two distinct slow-warming protocols, both lasting 2 minutes:
  1. 37°C: Samples warmed in CaniPlus AI medium at 37°C.
  2. 42°C: Samples warmed in CaniPlus AI medium at 42°C.
• Evaluation: Sperm quality was assessed before vitrification and after warming using Computer-Assisted Sperm Analysis (CASA) for motility and kinetic parameters (VCL, VSL, VAP, LIN, STR, ALH, BCF), SpermBlue staining for morphology, and eosin-nigrosine staining for viability. Statistical analysis employed repeated-measures ANOVA with Tukey's post hoc test.

Key Results
The study demonstrated that while vitrification significantly reduced sperm quality compared to fresh samples, the warming temperature critically influenced post-thaw recovery:

• Viability and Motility: Warming at 37°C resulted in significantly higher total motility (40.13% vs. 24.33%) and viability (47.53% vs. 30.23%) compared to warming at 42°C (p < 0.05). Progressive motility was numerically higher at 37°C but did not reach statistical significance between the two warming groups.
• Morphology: The percentage of morphologically normal spermatozoa was significantly higher in the 37°C group (72.91%) compared to the 42°C group (68.51%). The most common abnormalities observed post-vitrification were cytoplasmic droplets and bent or coiled tails.
• Kinetic Parameters: Most velocity parameters (VCL, VSL, VAP, LIN, ALH, BCF) showed no significant differences between fresh and vitrified samples, nor between the two warming regimens. However, straightness (STR) did show significant variation.
• Extender Performance: The coconut water and egg yolk combination successfully supported the vitrification process, yielding post-warming parameters comparable to or exceeding previously reported values for epididymal sperm.

Significance and Claims
The authors conclude that the warming protocol is a decisive factor in the success of vitrifying canine epididymal spermatozoa. Specifically, the study claims that warming at 37°C for 2 minutes provides superior preservation of sperm function (viability, total motility, and normal morphology) compared to warming at 42°C.

This finding contrasts with some previous data on ejaculated semen, suggesting that the unique biological characteristics of epididymal sperm (such as the absence of seminal plasma proteins) may make them more sensitive to higher warming temperatures. The paper posits that the combination of a coconut water-based extender with egg yolk, coupled with a 37°C warming regimen, represents a practical and effective approach for canine germplasm banking. The study emphasizes that optimizing warming conditions is a critical component of vitrification protocols for preserving valuable genetic resources, particularly for animals where epididymal recovery is the only option for genetic preservation. The authors note limitations regarding sample size and call for future research to evaluate fertility outcomes via artificial insemination and to explore additional cryoprotective additives.