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Determinations in Nanomedicine & Nanotechnology

Effect of refreezing on the plasma membrane integrity, acrosome, viability, morphology and individual motility in bovine sperm

Submission: February 19, 2021;Published: March 25, 2021


The purpose of this study was to evaluate the effect of refreezing of bovine semen, on the individual motility, viability, morphology, Animal Reproduction Laboratory in Zamorano University, Honduras. The semen of a 60-month-old Brahman and integrity of the sperm’s plasma membrane. The research was carried out from November 2018 to September 2019 at the bull, which was collected twice. The electro-ejaculation methodology was applied for the semen collection. Three treatments were applied: Fresh-Semen Diluted (FSD), Frozen-Thawed Semen (FTS) and Frozen-Thawed-Refreezing Semen (FTRS). In fresh semen the ejaculate volume (7mL), color (creamy white), odor (typical of the species), mass motility (>90%), individual sperm motility (>90%), concentration (739 x106/mL) and morphology (>90% normal) were evaluated. Subsequently, the semen was diluted using a TRIS- based diluent, citric acid, sugar, egg yolk, buffers, glycerin, double-distilled water, and the following antibiotics: tylosin, gentamicin, spectinomycin and lincomycin. A sample of the FSD was taken and evaluated for: individual motility, viability, morphology, and Hypo-Osmotic-Swelling-Test (HOST). The remaining diluted semen was packed in 0.5cc straws at a concentration of 30x106 sperm/straw, and frozen at -196 °C in liquid nitrogen. After five days, 20 straws were randomly taken and subjected to the FTS treatment, which consisted of thawing the straws at 38 °C/45 seconds, using water bath and further evaluation while another 20 straws were put through to the FTRS treatment, which consisted of defrosting the straws at 38 °C/45 seconds in a water bath. Subsequently they were left at room temperature for 60 minutes, and then were re-frozen, placing them at 4 °C for four hours (equilibrium) and then subjected to liquid nitrogen vapors for 10 minutes after that they were introduced in liquid nitrogen -196 °C. After 24 hours, six straws were thawed at 38 °C/45 seconds to be re-evaluated. The analyzed variables were individual motility (%), biological quality (individual post-frozen motility per concentration. The results must be greater than 10.5 x 106 sperm to be considered as a suitable sample for artificial insemination); Viability (% alive and dead using the BLOOM technique); Morphology (% normal sperm using Spermac® staining); Integrity of the plasma membrane using the Hypo-Osmotic-Swelling-Test (HOST): percentage (%) of Positive Endosmosis (PE) that corresponds to sperm with different degrees of curl/swelling of the tail and Negative Endosmosis (NE) that corresponds to the sperm without reaction in the tail. For the viability, morphology and membrane integrity tests (HOST test), at least 1200 sperm were counted in each one. In the individual motility and biological quality tests, at least 10 optical fields per test were evaluated. A Completely Randomized Design (CRD) was used with three treatments (FSD, FTS, FTRS) and two repetitions per treatment. The arc-sin function was applied for the conversion of percentage values and an Analysis of Variance (ANOVA) using the General Linear Model (GLM) and separation of means with the Duncan multiple range test, using the Statistical Analysis Systems program (SAS) with a required level of significance of P≤0.05. In semen FSD, FTS and FTRS the percentages of individual motility were 92.5%; 65.42% and 46.25% respectively (P≤0.05); the percentages of live sperm (P≤0.05) were 84.18%; 61.70% and 48.21% respectively and the percentages of normal sperm were 97.6%; 95.97% and 93.46% respectively. Regarding the HOST tests, the values were 82.5%; 50.13% and 31.58% for FSD, FTS and FTRS respectively (P≤0.05). The biological quality was 19.6 and 13.8 x 106 for FSD and FTRS respectively (P≤0.05). A high correlation (P<0.0001) was found between Positive Endosmosis with individual motility, biological quality, normality, and viability. The findings indicate that the freezing, thawing and refreezing process, affect the plasma membrane integrity, resulting in a higher percentage of sperm with Positive Endosmosis the FSD treatment. However, a decrease in FTS and FTRS was observed, both treatments showed higher values than the ones found in other studies. The values of individual motility, viability, normality, and biological quality were affected by the process of freezing and refreezing, nevertheless, all these exceed the established ranges as seminal samples suitable to be used in artificial insemination. These results suggest that the refreezing technique FTRS is viable in bovine semen.

Keywords:Ejaculate; Endosmosis, Morphology; Motility; Plasma membrane; Viability

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