Effects of protein supplements on fertility and assessment of the fertility genes (GDF9 and BMP15) in indigenous sheep of Bangladesh

Figures & data

Table 1. Feed ingredients and chemical composition of experimental ration.

Treatment groups	Ingredients											Calculated chemicals composition
	Maize	Rice Polish	Wheat bran	Soybean oil	Soybean meal	Protein concentrate	Dicalcium phosphate	DL-Methionine	Vitamin B premix	Common salt	Total amount
												Total Crude Protein (%)	Total Crude Fibre (%)	Ether Extract (%)	Total Energy, ME (MJ)
Control (T0)	59	22	10	0.75	5	0	1.15	0.6	1	0.5	100	11.68	4.44	6.03	12.00
Treatment 1 (T1)	56.5	22	10	0.75	5	2.5	1.15	0.6	1	0.5	100	12.94	4.46	6.19	12.00
Treatment 2 (T2)	54.5	22	10	0.75	5	4.5	1.15	0.6	1	0.5	100	13.96	4.48	6.32	12.00

Table 2. General characteristics of investigated genes (BMP15 and GDF9) (Hanrahan et al. 2004; Maitra et al. 2016).

	BMP15	GDF9
Primer Sequence	Forward Primer-5′-TCCCTAAAGGCCTGAAAGAGT-3′ Reverse Prime-5′-GCTGAAGGCAAGGAATAGAATC-3′	Forward Primer-5′-GAAGACTGGTATGGGGAAATG -3′ Reverse Prime-5′- CCAATCTGCTCCTACACACCT-3′
Amplified region	Exon 2	Exon 1
PCR Product(bp)	575	462
Annealing temperature°C	56	60

Table 3. Mean ± standard error of fertility traits of indigenous ewes of Bangladesh.

Parameters	Control			Average	Treatment 1			Average	Treatment 2			Average
	1–2 years (N = 3)	>2–3 Years (N = 3)	>3–5 years (N = 3)		1–2 years (N = 4)	>2–3 Years (N = 6)	>3–5 years (N = 6)		1–2 years (N = 3)	>2–3 Years (N = 6)	>3–5 years (N = 2)
CR %	66.67 (2)	66.67 (2)	66.67 (2)	66.67^b	75 (3)	83.33 (5)	83.33 (5)	80.55^a ± 2.78	66.67 (2)	83.33 (5)	100.00 (2)	83.33^a ± 9.62
GP (days)	153.50^b ± 0.50 (2)	155.50^a ± 0.50 (2)	153.00^b ± 1.00 (2)	154.00^a ± 0.67	152.67^a ± 0.88 (3)	150.60^b ± 0.40 (5)	150.00^b ± 0.71(5)	151.09^b ± 0.45	152.00^a ± 1.00 (2)	150.00^b ± 0.71 (5)	150.50^ab ± 0.50 (2)	150.83^b ± 0.50
LI (days)	183.50^b ± 1.50 (2)	188.00^a ± 2.00 (2)	186.50^a ± 2.50 (2)	186.00^a ± 1.24	183.33^b ± 1.20 (3)	189.00^a ± 1.87 (5)	185.00^b ± 1.48 (5)	185.77^a ± 1.18	178.50^a ± 2.50 (2)	172.40^b ± 1.08 (5)	172.50^b ± 1.50 (2)	174.46^b ± 1.16
LS (No)	1.50 ± 0.50 (2)	2.00 (2)	1.50 ± 0.50 (2)	1.67 ± 0.21	1.33 ± 0.33 (3)	1.60 ± 0.25 (5)	1.40 ± 0.25 (5)	1.44 ± 0.14	1.50 ± 0.50 (2)	1.60 ± 0.25 (5)	2.00 (2)	1.70 ± 0.17
BW (kg)	1.07^b ± 0.03 (3)	1.14^b ± 0.01(4)	1.29^a ± 0.02 (3)	1.17^b ± 0.03	1.20^b ± 0.01 (5)	1.22^b ± 0.02 (9)	1.31^a ± 0.02 (6)	1.24^b ± 0.01	1.32^b ± 0.05 (3)	1.46^a ± 0.11 (8)	1.65^a ± 0.05 (4)	1.48^a ± 0.07

Means with different superscripts in the among treatment and age group in treatment differ significantly (P < 0.05).

CR = conception rate; GP = gestation period; LI = lambing interval; LS = Litter size; BW = Birth weight; N is the number of ewe in each age group; parenthesis indicate the number of observation.

Figure 1. (a) PCR products of BMP15 gene 575 bp amplicon size; (b) PCR products of BMP15 gene 575 bp amplicon size.

Figure 2. (a) PCR products of GDF9 gene 462 bp amplicon sizes; (b) PCR products of GDF9 gene 462 bp amplicon sizes.

Figure 3. (a) The 100% identity sequence of OvisariesGDF9 genes (sequence ID: HE866499.1); (b) The 100% identity sequence of OvisariesBMP15 gene (sequence ID: KT853038.1).

Figure 4. (a) Nucleotide sequences alignment of GDF9 gene (Control = G1 DNA_GDF9 & G2 DNA_GDF9; Treatment 1 = G3 DNA_GDF9, G4 DNA_GDF9 & G5 DNA_GDF9; and Treatment 2 = G6 DNA_GDF9, G7 DNA_GDF9 & G9 DNA_GDF9) (b) Nucleotide sequences alignment of BMP15 gene (Control = B1DNA_BMP15; Treatment 1 = B4DNA_BMP15 & B5DNA_BMP15; and Treatment 2 = B7 DNA_BMP15)

Figure 5. (a) Phylogenetic tree drawn based on nucleotide sequences of the BMP15gene Legends: KT013294.1 = Ovis aries breed Lori Bakhtiari, NM001114767.1 = Ovis aries bone morphogenetic protein 15 (BMP15), mRNA,AH009593.2 = Ovis aries chromosome X growth differentiation factor 9B precursor,gene, complete cds, KT853038.1 = Ovis aries breed Pelibuey bone morphogenetic protein 15 (BMP15)mRNA, complete cds,B1DNA BMP15-R = Indigenous local sheep of Bangladesh,JN655671.1 = Ovis aries breed Lohi bone morphogenetic protein 15 (BMP15) gene,complete cds, HM583333.1 = Ovis aries isolate MS11R bone morphogenetic protein 15 (BMP15)gene, exon 2 and partial cds. (b) Phylogenetic tree drawn based on nucleotide sequences of the GDF9 gene Legends: KT357483.1 = Ovis aries breed Awassi (GDF-9)gene, partial cds, KT357482.1 = Ovis aries breed Barki(GDF-9)gene, partial cds, KT357485.1 = Ovis aries breed Suffolk X Awassi (GDF-9) gene, partial cds, KT357486.1 = Ovis aries breed Ossimi(GDF-9)gene, partial cds, KT357481.1 = Ovis aries breed Rahmani (GDF-9)gene, partial cds, G1DNAGD-F = Indigenous local sheep of Bangladesh, HE866499.1 = Ovis aries GDF9 gene, breed Norwegian white sheep, AF078545.2 = Ovis aries GDF-9 gene, complete cds, KT853039.1 = Ovis aries breed Pelibuey, KX377509.1 = Ovis aries breed Mehraban (GDF9)gene, exon 1 and partial cds.

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