Caffeine and oocyte vitrification: Sheep as an animal model

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

Figures & data

ig. 1 Representative images of spindle (green) morphology and chromatin (blue) alignment in ovine oocytes following immunostaining at 24 h.p.m. (A–C) show normal morphology (symmetrical barrel shape meiotic spindle with chromatin aligned regularly along the equatorial plane of spindle), while (D–F) illustrates abnormal morphology in vitrified, toxicity and control oocytes, respectively. Scale bar = 50 µm.

able 1 Effect of caffeine treatment during IVM on spindle and chromatin configuration of ovine oocytes vitrified at GV-stage.

Treatments	Caffeine	No. Oocytes	Spindle No. (%)			Chromosome No. (%)
			Normal	Abnormal	Missing	Normal	Abnormal
Vitrified	+	46	22 (47.8)^ab	11 (23.9)^a	13 (28.3)^a	22 (47.8)^ab	24 (52.2)^a
	–	57	25 (43.8)^a	16 (28.1)^a	16 (28.1)^a	25 (43.8)^a	32 (56.1)^a
Toxicity	+	44	23 (52.3)^ab	11 (25.0)^a	10 (22.7)^ac	23 (52.3)^ab	21 (47.7)^ac
	–	46	21 (45.7)^a	13 (28.3)^a	12 (26.1)^a	21 (45.7)^a	25 (54.3)^a
Control	+	44	34 (77.3)^b	6 (13.6)^a	4 (9.1)^bc	34 (77.3)^b	10 (22.7)^b
	–	102	69 (67.6)^ab	26 (25.5)^a	7 (6.8)^b	69 (67.6)^ab	33 (32.4)^bc

Unless indicated otherwise, data show the number of oocytes at each category, with percentages given in parentheses. + means oocytes were incubated in maturation medium with 10 mM caffeine (18–24 h.p.m.), while; − means oocytes matured in medium without caffeine. Values with different superscripts in the same column differ significantly (P ≤ 0.05).

ig. 2 Effects of caffeine supplementation during IVM on maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities of ovine oocytes vitrified at the germinal vesicle stage. Oocytes from control, toxicity and vitrified groups were incubated in IVM medium either supplemented with + or without − caffeine (10 mM) at 18–24 h.p.m., at 24 h.p.m., MPF and MAPK activities were detected in oocytes with an in vitro double-kinase by phosphorylation of histone H1 and myelin basic protein (MBP), respectively. A) Relative kinase activities (cpm/mm²). B) Phospho-image of PAGE, ³²P radioactivity representing kinase activities. Ten oocytes were analyzed in each group and three replicates were repeated for each experimental group. Data are presented as means ± SEM.

ig. 3 Effects of caffeine supplementation during IVM of ovine oocytes vitrified at the germinal vesicle stage on mean cell numbers of blastocysts produced after IVF and embryo culture. Mean cell numbers per blastocyst were determined by staining of day 7 blastocysts with Hoechst 33342 (10 µg/mL). Data are presented as means ± SEM. Bars with different letters denote significant differences at (P ≤ 0.05).

able 2 Effect of caffeine treatment during IVM on developmental potential of ovine oocytes vitrified at the germinal vesicle stage.

Treatment	Caffeine	No. Oocytes	No. Cleaved (%)		No. Morula (%)	No. Blastocysts (% of oocytes)				Total blastocysts
			24 h.p.i.	48 h.p.i.		Early	Expanded	Hatched	Total	(% cleaved at 48 h.p.i.)
Vitrified	+	142	4 (2.8)^a	43 (30.3)^a	36 (25.4)^a	5 (3.5)^ac	1 (0.7)^a	0 (0.0)^a	6 (4.2)^a	13.9^a
	−	115	5 (4.3)^a	37 (32.2)^a	28 (24.3)^a	0 (0.0)^c	2(1.7)^ac	0 (0.0)^a	2 (1.7)^a	5.4^a
Toxicity	+	98	31 (31.6)^b	77 (78.6)^b	53 (45.1)^bc	9 (9.2)^a	10 (10.2)^b	12 (12.2)^b	31 (31.6)^b	40.3^b
	−	93	21 (22.6)^b	54 (58.1)^b	33 (35.5)^ac	3 (3.2)^ab	12 (12.9)^b	15 (16.1)^b	30 (32.2)^b	55.5^b
Control	+	149	47 (31.5)^b	94 (63.1)^b	62 (41.6)^bc	11 (7.4)^ab	10 (6.7)^bc	24 (16.1)^b	45 (30.2)^b	47.9^b
	−	96	52 (45.2)^c	67 (69.8)^b	37 (38.5)^ac	10 (10.4)^b	11 (11.5)^b	8 (8.3)^b	29 (30.2)^b	43.3^b

Cleavage rates were evaluated at 24 and 48 h post insemination (h.p.i.). Morula and blastocyst rats were evaluated on Days 5 and 7 post insemination, respectively. Unless indicated otherwise, data show the number of oocytes at each stage, with percentages given in parentheses. + means oocytes were incubated in maturation medium supplemented with 10 mM caffeine (18–24 h.p.m.), while; − means oocytes matured in medium without caffeine. Values with different superscripts in the same column differ significantly (P ≤ 0.05).