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. 2021 Aug;100(8):101291.
doi: 10.1016/j.psj.2021.101291. Epub 2021 May 27.

Type III antifreeze protein (AFP) improves the post-thaw quality and in vivo fertility of rooster spermatozoa

Mahdieh Mehdipour  1 , Hossein Daghigh-Kia  2 , Abouzar Najafi  3 , Felipe Martínez-Pastor  4
Affiliations

Type III antifreeze protein (AFP) improves the post-thaw quality and in vivo fertility of rooster spermatozoa

Mahdieh Mehdipour et al. Poult Sci. 2021 Aug.

Abstract

Antifreeze proteins (AFP) have the potential for improving sperm cryopreservation. We have applied Type III antifreeze protein (AFP3) on the cryopreservation of spermatozoa from broiler breeder roosters, aiming to enhance post-thawing quality and fertility. Semen was extended at 37°C in Lake's extender containing AFP3 at 0.01, 0.1, 1, 5, and 10 µg/mL (no AFP3 as control). Post-thawing sperm assessment included sperm motility (CASA), morphology, membrane functionality by hypoosmotic swelling test (HOST), lipoperoxidation as malondialdehyde (MDA) production, and sperm viability, early apoptosis (phosphatidylserine exposure as annexin V-positive staining in viable spermatozoa), and mitochondrial activity by flow cytometry. Fertility was assessed after artificial insemination (30 hens/treatment). Total and progressive motility, membrane functionality, and mitochondrial activity increased in 0.1 and 1 µg/mL AFP, compared to control and other concentrations, whereas apoptosis was significantly lower. VAP, VSL, and viability were significantly higher for 1 µg/mL AFP3 than with the other treatments except for 0.1 µg/mL (which was not always significantly different from the control or other concentrations), and with abnormal forms being significantly lower. The proportion of fertilized and hatched eggs was also higher for 1 µg/mL AFP3, with 0.1 µg/mL also showing significantly higher results than the control, and no differences with other concentrations). In conclusion, 1 µg/mL AFP3 could improve the post-thawing results of rooster spermatozoa frozen in Lake's extender. According to our results, concentrations between 1 and 0.1 µg/mL could be similarly efficient.

Keywords: cryopreservation; rooster, spermatozoon; type III antifreeze protein.

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Figures

Figure 1
Figure 1
Examples of spermatozoa as evaluated for the hypo-osmotic swelling (HOS) test for membrane functionality assessment. The swollen spermatozoa are shown by arrow.
Figure 2
Figure 2
Results for CASA parameters after cryopreserving rooster semen with different Type III AFP concentrations. The figures show mean ± 95% CI for control (0 µg/mL) and Type III AFP treatments, with observations as dots. Treatments with different Latin lowercase letters differ P ≤ 0.05. Abbreviations: ALH, mean amplitude of the lateral head displacement (µm); BCF, mean of the beat cross frequency (Hz); LIN, linearity (%); STR, straightness (%); VAP, average path velocity (µm/s); VCL, curvilinear velocity (µm/s); VSL, straight-line velocity (µm/s); WOB, Wobble (%);.
Figure 3
Figure 3
Results for HOS test, abnormal forms, and physiological parameters assessed by flow cytometry after cryopreserving rooster semen with different Type III AFP concentrations. The figures show mean ± 95% CI for control (0 µg/mL) and Type III AFP treatments, with observations as dots. Treatments with different Latin lowercase letters differ P ≤ 0.05. The ratio refers to the proportion of positive spermatozoa within the viable (PI–) population. Abbreviations: Act. mit., active mitochondria; Apop. ratio, apoptotic ratio.
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