Evaluation of bull spermatozoa during and after cryopreservation: Structural and ultrastructural insights

Peer review under responsibility of Faculty of Veterinary Medicine, Cairo University.

Figures & data

Fig. 1 Representative images of scanning electron microscopy showing examples of sperm cell abnormalities which occur during cryopreservation of bull semen. A: Detached and cracked head, B: coiled tail.

Table 1 The effect of different processing steps of semen cryopreservation on sperm motility, viability and morphological abnormalities examined by light microscopy (mean percentage ± SEM, n = 5).

	Motility	Dead	Abnormal
Raw semen	77 ± 1.7^a	23 ± 0.7^a	19 ± 0.8^a
Freshly diluted semen (37°C)	71 ± 0.8^a	26 ± 0.4^a	20 ± 1.2^a
Cooled semen (4°C, 2 h)	67 ± 1.1^b	30 ± 1.6^b	22 ± 1.0^b
Equilibrated semen (4°C, 4 h)	63 ± 1.7^b	35 ± 1.3^c	24 ± 0.5^c
Frozen-thawed semen	50.8 ± 2.7^c	45 ± 2.2^d	29 ± 1.5^d

Different superscripts indicate significant differences at P < .05 within each column.

Fig. 2 Representative images of sperm head showing patterns of plasma membrane integrity (arrows) at different stages of semen processing and cryopreservation. A: Intact plasma membrane in fresh semen sample. B: Slightly swollen PM in diluted semen, C: Swollen undulating PM in frozen thawed sample. Cross sections of sperm cells in A and C show the same PM integrity patterns.

Table 2 The effect of different processing steps of semen cryopreservation on sperm plasma membrane (PM) and acrosome reaction (AR) examined by transmission electron microscopy.

Sample	Slightly swollen PM			Swollen PM			Lost PM
	P%	OR	CI (95%)	P%	OR	CI (95%)	P%	OR	CI (95%)
Diluted (37°C)	16^a			2^a			2^a
Cooled (4°C, 2 h)	21^a	1.40	0.68–2.87	2^a	1.00	0.14–7.24	2^a	1.00	0.14–7.24
Equilibrated (4°C, 4 h)	22^a	1.48	0.73–3.02	15^b	8.65	1.92–38.90	3^a	1.52	0.25–9.27
Frozen-thawed (37°C)	10 ^a	0.58	0.25–1.36	40^c	32.67	7.62–140.1	10^b	5.44	1.16–25.52
Sample	Typical AR			Atypical AR			Lost acrosome
	P%	Exact Sig.^*		P%	OR	CI (95%)	P%	OR	CI (95%)
Diluted (37°C)	0^a			9^a			1^a
Cooled (4°C, 2 h)	0^a	1.000		14^a	1.64	0.67–3.99	1^a	1.00	0.62–16.2
Equilibrated (4°C, 4 h)	3^a	0.246		16^a	1.93	0.81–4.59	1^a	1.00	0.62–16.2
Frozen-thawed (37°C)	10^b	0.002		19^b	2.37	1.06–5.54	6^a	6.32	0.74–53.5

Data are shown as the “P” proportions of each parameter per 100 sperm counted in each sample. OR: Odds ratio and CI (95%): confidence interval are shown in comparison to freshly diluted semen at 37°C. Different alphabet superscripts within each column indicate significant different at P < .05 as analyzed by logistic regression (Wald test).

* Typical AR was analyzed using 2-tailed Fisher exact test because the probability of 2 groups equals zero.

Fig. 3 Representative micrographs showing ultrastructural changes in sperm acrosome. ACS1: Intact acrosome, ACS2: typical acrosome reaction (AR) and ACS3: Atypical AR.

Fig. 4 Mitochondrial morphology and damage induced by cryopreservation (arrows). Longitudinal Ultrathin section of raw semen showing a sperm mid piece. A: normal mitochondria; B and C: damaged mitochondria with distorted cristae. Magnification 10,000×.

Table 3 The effect of different processing steps of semen cryopreservation on sperm mitochondrial damage examined by transmission electron microscopy.

Sample	Mitochondrial damage
	P%	OR	CI (95%)
Diluted (37°C)	2^a
Cooled (4°C, 2 h)	2^a	1.00	0.14–7.24
Equilibrated (4°C, 4 h)	6^a	3.13	0.62–15.89
Frozen-thawed (37°C)	15^b	8.65	1.92–38.9

Data are shown as the “P” proportions of each parameter per 100 sperm counted in each sample. OR: Odds ratio and CI (95%): confidence interval are shown in comparison to freshly diluted semen at 37°C. Different alphabet superscripts within each column indicate significant difference at P < .05 as analyzed by logistic regression (Wald test).

Fig. 5 Nuclear morphology and damage induced by cryopreservation. Longitudinal Ultrathin section of the head piece showing abnormal nucleus (N, right) with big nuclear vacuole containing electron dense material and indicating chromatin damage compared to normal (left) nucleus with homogenous condensed chromatin.

Table 4 The effect of different processing steps of semen cryopreservation on sperm DNA damage examined by transmission electron microscopy, TB staining and SCSA.

Sample	Chromatin damage assessed by TEM			Chromatin damage accessed by SCSA			Chromatin damage assessed by TB
	P%^*	OR	CI (95%)	P%^*	OR	CI (95%)	P%^*	OR	CI (95%)
Diluted (37°C)	3			2.8^a			1.3^a
Cooled (4°C, 2 h)	3	1.00	0.20–5.08	3.8^b	1.36	1.16–1.59	2.0^a	1.51	0.42–5.41
Equilibrated (4°C, 4 h)	4	1.35	0.29–6.18	4.8^c	1.79	1.54–2.08	2.7^a	2.02	0.60–6.81
Frozen-thawed (37°C)	9^§	3.20	0.84–12.18	8.4^d	3.22	2.79–3.69	4.7^b	3.62	1.18–11.14

* Data are shown as proportions evaluated by TEM (in a total of 100 sperm per sample), microscopically following TB staining (counting per 300 sperm per sample), or evaluated by flowcytometry following SCSA (in 10,000 sperm per sample). The sign § indicates tendency to increase nuclear damage; P = .089. Different superscripts within the same column indicate significant difference analyzed by logistic regression (Wald test). OR: Odds ratio, CI: confidence interval.