Effects of selenium deficiency on testis development and autophagy in chicks

Figures & data

Table 1. Composition of basal diet fed to chicks.

Ingredient	Basal diet (% of diet as fed basis)
Ground corn^a	53.825
Soybean meal^a 48%	29.45
Ground limestone	10.5
Corn oil	4.10
Dicalcium phosphate	0.985
Iodized salt	0.475
Trace mineral mix^b	0.25
Vitamin mix^c	0.20
DL-methionine	0.215
Calculated composition^d	–
MEn, Mcal/kg^e	2.90
Protein, %	18.90
Calcium, %	4.26
Available P, %	0.30
Methionine, %	0.52
TSAA^f, %	0.82
Lysine, %	1.04

^a Corn and soy were produced in the Se-deficient region of Heilongjiang Province, China, and the diet contained 0.033 mg of Se/kg in low-Se group.

^b Provided the following per kilogram of diet: 0.333 mg selenium, 6 mg copper, 0.53 mg iodine, 120 mg iron, 83 mg manganese, 60 mg zinc and 5 mg cobalt. No selenium was provided by the mineral mix in low-Se group.

^c Provided the following per kilogram of diet: 7,937 IU vitamin A, 2,205 IU vitamin D3, 26 IU vitamin E, 1.6 mg vitamin K, 1.6 mg thiamine, 5.3 riboflavin, 8.8 mg pantothenic acid, 35 mg niacin, 3.2 mg pyridoxine, 0.088 mg biotin, 1.1 mg folic acid, 0.016 mg vitamin B12, and 397 mg choline.

^d Values reported on an as-fed basis.

^e ME_n: metablic energy.

^f Total sulphur amino acids (methionine + cysteine).

Table 2. Gene-special primers for Beclin-1, Dynein, ATG-4B, ATG-5, LC3-A, LC3-B and β-actin used in the qPCR.

Gene	Primer sequence	Primer length (bp)
Beclin-1	F: 5′-CAGACACGCTGCTGGACC-3′	18
	R: 5′-TCTCCTTGTCATCCTCGTTCA-3′	21
Dynein	F: 5′-TGGGATAATCGCAGCAATAAGA-3′	22
	R: 5′-AGGGAAGGACATGCAAGTAACAG-3′	23
ATG-4B	F: 5′-GAGACTGGAGGTTAAAAGG-3′	22
	R: 5′-ATCTGGGCTATCTGGTGAATG-3′	21
ATG-5	F: 5′-GATGAAATAACTGAAAGGGAAGC-3′	23
	R: 5′-TGAAGATCAAAGAGCAAACCAA-3′	22
LC3-A	F: 5′-GCATCCAAACAAAATCCCAGTC-3′	22
	R: 5′-AAGCCATCCTCATCCTTCTCCT-3′	22
LC3-B	F: 5′-CTTCTTCCTCCTGGTGAACG-3′	20
	R: 5′-GCACTCCGAAAGTCTCCTGA-3′	20
β-actin	F: 5′-CACCACAGCCGAGAGAGAAAT-3′	21
	R: 5′-TGACCATCAGGGAGTTCATAGC-3′	22

Figure 1. Effects of Se-deficiency on the weight and male organ coefficient of chicks. (A) body weight, (B) testicle weight and (C) organ coefficient. ***Significant differences (p < .001) between the CK group and the low Se group. ns: no significant differences.

Figure 2. Photomicrographs of chick testis (HE staining). (A) The normal architecture of testis tissue in the control group (400×). (B, C) Testis tissue was injured in the Low Se group (400×). (D) Testis tissue was injured in the low Se group (200×).

Figure 3. Effects of Se-deficiency on changes of serum sex hormones and antioxidant enzymes. (A) Testosterone, (B) Oestradiol, (C) T-AOC, (D) GSH-ST and (E) GSH-PX. **, ***Significant differences (p < .01, p < .001) between the CK group and the low Se group.

Figure 4. Effects of Se-deficiency on autophagy-related factors mRNA expression in chick testis. The mRNA expression of (A) Beclin1, (B) Dynein, (C) ATG-4B, (D) ATG5, (E) LC3-A and (F) LC3-B genes, respectively. In the experiment, the relative mRNA expression of the autophagy genes was detected by qPCR. *, **Significant differences (p < .05, p < .01) between the CK group and the low-Se group.

Figure 5. Effects of Se-deficiency on autophagy-related factors protein expression in chick testis. The protein expression of (B) Beclin1, (C) Dynein, (D) ATG5 and (E) LC3-B genes, respectively. In the experiment, the relative protein expression of autophagy genes was detected by western blot. *Significant differences (p < .05) between the CK group and the low Se group.