The Acrosome Integrity Examination of Post-thawed Spermatozoa of Several Ongole Grade Bull in Indonesia Using Giemsa Staining Method

Abstract

Cement cryopreservation is a preserving technique of spermatozoa cells that the spermatozoa cells can be used in a long period of time. Frozen semen is produced by the National Artificial Insemination Center (AIC) and the Regional Artificial Insemination Center (RAIC) in Indonesia. The indicators in predicting spermatozoa fertilizing ability include individual motility and movement scores, but the indicators have not been able to accurately predict the spermatozoa fertilizing ability, Acrosome integrity of spermatozoa cells is an important indicator of the success of the fertilization. The study aimed at evaluating the quality of the acrosome integrity of Ongole Grade Bull post-thawed spermatozoa from several Artificial Insemination Centers (AIC) in Indonesia using Giemsa staining method. It used 60 straw samples of 6 PO cattle aged 6 to 8 years with healthy and normal reproductive organs obtained from 3 different Artificial Insemination Centers (AICs) in Indonesia. The spermatozoa acrosome integrity was evaluated using Trypan Blue-Giemsa (TBG) and Giemsa stainings combined with fixation using methanol. The results showed that the motility (43.60±1.65%) and the viability of the spermatozoa (55.75±1.83%) of Ongole grade bull in the AIC A was higher than those in the other AIC (P<0.05). The results of the evaluation of the acrosome integrity of the Ongole grade bull from the AIC A using the TBG staining showed that the acrosome integrity of the bull from the AIC A was higher than that of the bull from the other AIC (P<0.05) (77.00±1.52%). They also showed that the acrosome integrity of the Ongole grade bull from the AIC A evaluated using Giemsa staining was also higher than that of the bull from the other AIC (P<0.05) (76.84±1.57%). Spermatozoa acrosomes can be evaluated using TBG or Giemsa stainings. However, the TBG staining is more complex and it takes a long time for the staining to give expected results. Meanwhile, the Giemsa staining combined with fixation using methanol is a simple method and it does not take a long time for the staining to give satisfying results.

Deterioration of Frozen Semen of Bali Cattle after Cooling at 5°C

“…Individual variations of Bali cattle caused significant differences in the intact acrosome cap values of AD, PT1, and PT2 groups (p < 0.05, Table 3). Before freezing, Bali cattle's average intact acrosome cap was 91.06% (Damayanti et al, 2021), while frozen semen was 64.12 ± 1.21-76.82 ± 1.55% (Prihantoko et al, 2020). The best percentage of intact acrosome cap in the PT1 group was for Individual E, with a value of 74.04%, but it was not different from individual J with a value of 73.89%.…”

“…The stained slide was washed with distilled water and observed using a microscope (Olympus CX23, Japan -Optilab advanced V2, Indonesia) by counting 200 spermatozoa. Acrosomes of intact sperm were indicated by the purple top of the head, while those with light color imply deterioration (Chowdhury et al, 2014;Prihantoko et al, 2020).…”

“…The distribution of tyrosine-phosphorylated acrosome protein from each male is different, resulting in individual differences in maintaining acrosome stability after the freezing process (Arai et al, 2017). Due to its small molecular weight, Giemsa staining can bind to proteins on the membrane and can pass through cell membranes that protect the acrosomes (Nofa et al, 2018;Prihantoko et al, 2020). Furthermore, the integrity acrosome is needed to ensure the success of spermatozoa in fertilizing the egg because the cap protects the enzymes contained (Sun et al, 2020).…”

Molecular Markers: A New Paradigm in the Prediction of Sperm Freezability

“…Even though eosin-nigrosin (EN) staining brings reliable results when identifying fresh semen quality, two studies [ 17 , 18 ] found this technique inappropriate when evaluating post-thaw spermatozoa. On the contrary, Trypan-blue/Giemsa (TBG) staining belongs to bright-field staining techniques with good credibility when analyzing the acrosomal damage in cryopreserved spermatozoa [ 19 ]. The acrosome intactness test (AIT) introduced by Gopalkrishnan et al [ 20 ] belongs to simple but tedious tests based on the halo effect of intact acrosomes in samples diluted with PBS and glucose solution smeared on gelatin-coated slides.…”

Evaluation of Kappa‐carrageenan supplementation in extender for post‐thaw Kajli ram sperm quality

“…Cryopreservation is a technique that allows for the preservation of biological samples at extremely low temperatures, which slows down the metabolic activities of the cells, thereby ensuring their preservation for more extended periods (Davis et al., 1963). This technique brought a breakthrough revolution in assisted reproduction and is indispensable for livestock (Prihantoko et al., 2020). Although hypothermic conditions during cryopreservation enable the long‐term storage of biological samples, ultra‐low temperatures lead to irreversible mechanical damage at the cell level (Khan & Ijaz, 2008).…”