Effects of intratesticular injection of hypertonic mannitol and saline on the quality of donkey sperm, indicators of oxidative stress and testicular tissue pathology

Abstract

Objectives: The aim of the present study was to examine donkey sperm quality after intratesticular injection of hypertonic mannitol (HM) and saline (HS).

Methods: Randomly assigned to five treatment groups were 15 adult male donkeys: (1) Control group (no treatment), (2) Surgery group (surgical castration for testosterone control), (3) NS group (normal saline intratesticular injection), (4) HS group (hypertonic saline), and (5) HM group. We injected 20 mL per testicle. We took 5 mL blood from all donkeys before injection. Castration was performed under general anesthesia 60 days later. Samples included blood and testicular tissue. Total motility (TM), progressive motility (PM), movementy features, DNA damage, morphology, viability, and plasma membrane functionality were evaluated. Hormone analyses, histomorphometric studies and oxidative stress indices including total antioxidant capacity (TAC), glutathione peroxidase (GPx), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and NADP+/NADPH were evaluated. Apoptosis, pyroptosis-related Bax, Caspase-1, GSDMD, and Bcl-2 expression were also assessed.

Results: In HS and HM groups, testosterone, epididymal sperm count, motility, viability, and plasma membrane functionality dropped while sperm DNA damage increased. HS and HM groups had significantly lower histomorphometric parameters, TAC, GPx, SOD, GSH, and Bcl-2 gene expression. MDA, NADP⁺/NADPH, Bax, Caspase-1, and GSDMD gene expression were substantially higher in the HS and HM groups than in the control group.

Conclusions: Toxic effects of hypertonic saline and mannitol on reproductive parameters were seen following, hence, they might be considered as a good chemical sterilizing treatment in donkeys.

Keywords: Apoptosis; Chemical sterilization; Donkey; Genes expression; Pyroptosis.

Fig. 1

A Intratesticular injection of normal saline, (B) Day 60 of normal saline group, (C) Intratesticular injection of mannitol, (D) Day 60 of mannitol group

Fig. 2

Sperm PM functionality. White arrows - the spermatozoa with straight tails (nonfunctional PM); Black arrows - the spermatozoa with coiled tails (functional PM). (400 ×)

Fig. 3

Sperm DNA damage. Yellow arrows - normal spermatozoa (green); White arrows - DNA damaged spermatozoa (yellow-red). (Acridine orange, 400×)

Fig. 4

Sperm viability. Black arrow - Viable spermatozoa (colorless); White arrows - Dead spermatozoa (red). (Eosin/nigrosine, 400×)

Fig. 5

A Total antioxidant capacity (TAC); (B) Glutathione peroxidase (GPx); (C) Superoxide dismutase (SOD); (D) Glutathione (GSH) activities; (E) Lipid peroxidation (MDA); (F) NADP⁺/NADPH of donkey testes after intratesticular injection of hypertonic mannitol and hypertonic saline (HTS) in different experimental groups. NS: normal saline; HM: hypertonic mannitol; HS: hypertonic saline. ^abDifferent superscripts within the same row demonstrate significant differences (p ≤ 0.05; Mean ± S.E.M.)

Fig. 6

The effects of different treatments on testicular histoarchitecture. Hematoxylin and eosin, 400×). NS: normal saline; HM: hypertonic mannitol; HS: hypertonic saline

Fig. 7

The mRNA levels of (A) Bax, (B) Bcl-2, (C) Caspase-1, and (D) GSDMD were assessed using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The density of Bax, Bcl-2, Caspase-1, and GSDMD mRNA levels in testicular tissue was measured by densitometry and normalized to the GAPDH mRNA expression level. NS: normal saline; HM: hypertonic mannitol; HS: hypertonic saline. ^abSignificant differences between groups are indicated by different superscripts (p ≤ 0.05; Mean ± S.E.M.)

Fig. 8

The effectiveness of intravenous injections of mannitol and HTS for chemical sterilization