Fig 1.
Total cell viability of neonatal lamb testis tissue immediately after castration (fresh), cryopreserved by slow freezing and vitrification.
Values represent mean ± SD of three donors (three replicates per donor). Lines above the error bars denote significance.
Fig 2.
Histological appearance of immature lamb testis tissue exposed to one of three conditions.
(A) control (fresh), (B) slow freezing and then thawing or (C) vitrification and then thawing (400x). Inset in each panel depicts the seminiferous cords (SC) at a higher magnification. Slow freezing preserved normal SC integrity similar to the fresh control, whereas vitrification caused disruptions, including evidence of shrinkage around the SC (*). G = gonocyte, S = Sertoli cell, M = peritubular myoid cell. Scale bar = 100 μm.
Fig 3.
Xenograft volume density traits for pieces of lamb testis transplanted fresh (control) versus after slow freezing and thawing versus vitrification and thawing.
For each assessed metric, data for the slow freezing group were no different (P>0.05) from the control. Within a trait, lines above the error bar denote significance.
Fig 4.
Relative percentage of seminiferous cords versus tubules in cross-sections of lamb testis pieces that were retained fresh versus those that were subjected to slow freezing and warming versus those that were vitrified and thawed, with all then xenografted into SCID mice.
Testis fragments exposed to slow freezing were no different from controls (P>0.05) in contrast to a preponderance of only unchanged seminiferous cords in the vitrified group (Panel A). Within a trait, lines above the error bar denote significance.
Fig 5.
Morphology of lamb testis pieces that were retained fresh (A) versus those subjected to slow freezing and thawing (B) versus those that were vitrified and thawed (C).
All then were xenografted into SCID mice before excision and evaluation at 17 wk. Sections were stained with hematoxylin-eosin. Each scale bar = 100 μm.
Fig 6.
Prevalence of advanced germ cell type per seminiferous cord/tubule cross-sections in xenografts that originally were fresh controls versus those subjected to slow freezing versus vitrification.
The latter treatment resulted in production of mostly spermatogonial cells and primary spermatocytes in contrast to slow freezing where few immature germ cells remained along with a higher proportion of more advanced cell types, including elongated spermatids. Within a trait, lines above the error bar denote significance.