Effect of the Booroola fecundity (FecB) gene on the reproductive performance of ewes under assisted reproduction

Journal Pre-proof Effect of the Booroola fecundity (FecB) gene on the reproductive performance of ewes under assisted reproduction

Mei-Yu Qi, Li-Qiang Xu, Jia-Nan Zhang, Meng-Ou Li, Ming-Hai Lu, Yu-Chang Yao PII:

S0093-691X(19)30492-3

DOI:

https://doi.org/10.1016/j.theriogenology.2019.10.038

Reference:

THE 15233

To appear in:

Theriogenology

Received Date:

30 April 2019

Accepted Date:

31 October 2019

Please cite this article as: Mei-Yu Qi, Li-Qiang Xu, Jia-Nan Zhang, Meng-Ou Li, Ming-Hai Lu, YuChang Yao, Effect of the Booroola fecundity (FecB) gene on the reproductive performance of ewes under assisted reproduction, Theriogenology (2019), https://doi.org/10.1016/j.theriogenology. 2019.10.038

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Journal Pre-proof Revised Effect of the Booroola fecundity (FecB) gene on the reproductive performance of ewes under assisted reproduction Mei-Yu Qi a,b, Li-Qiang Xu a,c, Jia-Nan Zhang a,c, Meng-Ou Li d, Ming-Hai Lu e, Yu-Chang Yao a,c* a

College of Animal Science and Technology, Northeast Agricultural University, Harbin, P.R.

China b

Animal Husbandry Research Institute, Heilongjiang Academy of Agricultural Sciences, Harbin,

P.R. China c

Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of

Heilongjiang province, Harbin, P.R. China d College e

of Life Science, Northeast Agricultural University, Harbin, P.R. China

Department of Animal Science, Heilongjiang State Farms Science Technology Vocational

College, Harbin, P.R. China

*Corresponding

author:

Yu-Chang Yao, College of Animal Science and Technology, Northeast Agricultural University, NO.600 Changjiang Street, Xiangfang District, Harbin, 150030, P.R. China. Email address:[email protected].

Telephone:+86-451-55190464.

1

Fax:

+86-451-55103336.

Journal Pre-proof ABSTRACT Reproductive traits are important factors in sheep production. The Booroola fecundity (FecB) gene—the first major gene for prolificacy identified in sheep—has a positive effect on ovulation rates and litter size under natural reproductive conditions. However, the effect of the FecB gene on reproductive performance under assisted reproduction, which uses many artificial hormones, remains unclear. In the present study, we evaluated the effect of FecB (BMPR-1B mutation) on reproductive performance under assisted reproduction, and examined offspring body weight at birth and weaning and survival rate at weaning. There were no differences among three genotype groups (homozygous carrier, BB; heterozygous carrier, B+; non-carrier, ++) in terms of estrus detection rate, time to estrus onset, or estrus duration following estrus synchronization (P>0.05). The pregnancy rates at 60 d were similar among three genotype groups after artificial insemination (P>0.05). However, the B allele had an additive effect on litter size (one copy resulted in an increase of 0.88 lambs and two copies produced an additional 0.41 lambs; P<0.01), and increased lambing and fecundity rates (P<0.01). After multiple ovulation, the average numbers of recovered embryos per ewe were 9.160.79, 8.200.77, and 8.440.61 in the BB, B+, and ++ ewes, respectively (P>0.05). There were no differences in the fertilization rate or numbers of grade 1-2 embryos among different groups (P>0.05). The birth and weaning weights of lambs from BB and B+ ewes were lower than those of lambs born from ++ ewes (P<0.01) owing to the high fecundity. The survival rate of lambs at weaning did not differ among groups (P>0.05). Our results indicated that the presence of the B allele had an additive effect on litter size after artificial insemination, but it did not influence the parameters of estrus synchronization and multiple ovulation. Furthermore, the higher prolificacy in ewes carrying the B allele was associated with a reduction in offspring body weight at birth and weaning. Keywords: Sheep; FecB gene; BMPR-1B mutation; Assisted reproductive technology; Reproductive

performance

2

Journal Pre-proof 1. Introduction Reproductive traits, such as ovulation rate and litter size, are important factors in sheep production. The Booroola fecundity gene (FecB) is the first major gene for prolificacy identified in sheep and it reportedly has an additive effect on ovulation rate and litter size in Booroola Merino sheep [1]. The effect of FecB is due to a mutation in the bone morphogenetic protein (BMP) receptor 1B (BMPR-1B), as reported by three independent groups [2-4]. The mutation is an A to G transition in exon 8 of the BMPR-1B gene, which results in a substitution of the 249th amino acid from glutamine to arginine (Q249R) in the highly conserved intracellular kinase signaling domain. This mutation can be detected directly using a forced PCR restriction fragment length polymorphism (RFLP) approach as described by Wilson et al. [4]. Considering the importance of this gene, many researchers began to screen other prolific sheep breeds to determine whether BMPR-1B mutation is responsible for their high prolificacy. The BMPR-1B mutation has been found in Garole and Javanese [5, 6], Hu and Small Tail Han (STH) [7-9], Kendrapada [10] and Kalehkoohi [11] sheep breeds. Under natural reproductive conditions, the effects of FecB on litter size, lambing performance, and other production traits have been widely reported [9, 12-14]. In recent years, studies have been conducted to investigate the mechanism by which FecB (BMPR-1B mutation) affects the ovulation rate and litter size in sheep [15, 16-19]. In STH sheep with all three FecB genotypes, the BMPR-1B gene was shown to be highly and widely expressed in many tissues, including the ovary and hypothalamus [15]. The FecB mutation altered the BMPR-1B functionality [20], increased response to bone morphogenetic protein (BMP) ligands in ovarian somatic cells [21], regulated the BMP/Smad signal pathway in the antral follicles [22], and reduced the mRNA level of BMP 15 in the oocytes [23] and ovaries [15]. Meanwhile, molecular marker assisted selection using this mutation has been applied to introduce FecB into some less prolific breeds in a global crossbreeding program aimed at improving fecundity [9, 24, 25]. Furthermore, Zhang et al. [26] reported that the BMPR-1B gene was disrupted by CRISPR/Cas9 in embryos to improve sheep reproductive traits. Assisted reproductive technology (ART) enhances animal reproductive efficiency and genetic improvement [27-29]. Traditionally, ARTs in sheep include estrus synchronization, artificial insemination (AI), and multiple ovulation and embryo transfer (MOET). Generally, the application of ARTs is combined with the use of many exogenous hormones, such as pregnant mare serum 3

Journal Pre-proof gonadotropin (PMSG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prostaglandin (PG), etc. Previous studies have shown no significant differences in the secretion patterns of pituitary gonadotropins or ovarian hormones between B allele carrier and non-carrier ewes [30, 31] and the FecB gene seems to exert control over follicle growth at the level of the ovary [30]. In this study, we hypothesize that FecB enhances ovarian sensitivity to hormone stimulation and improve ewe reproductive performance under ARTs. The objective of this study was to evaluate the effects of FecB (BMPR-1B mutation) on (1) reproductive performance following estrus synchronization, AI, and multiple ovulation, and (2) on the body weight and survival of offspring. Our research will provide additional information for the application of FecB.

2. Materials and Methods 2.1 Ethics statement All sheep were managed under normal husbandry conditions. All experimental animal protocols were approved and performed in accordance with the requirements of the Animal Care and Use Committee at Northeast Agricultural University (approval ID 2016–031). All surgeries were performed under sodium pentobarbital anesthesia and all efforts were made to minimize any suffering experience by the animals used in this study. 2.2. Animals The study was conducted in Daqing city, northeast China (46°33′ N, 124°49′ E), at an altitude of 150 m above mean sea level. A total of 231 reproductive healthy STH ewes (3 to 4 years of age) were used in this study. Peripheral blood samples were collected from the jugular vein of each ewe and immediately transported to the laboratory on ice before DNA isolation. All animals were raised under semi-intensive management system and provided with similar grazing conditions. In addition to grazing, the animals were provided with concentrate mixture (250 g/day). The lambs were allowed to suckle their dams from birth until weaning at 2 months of age. 2.3 DNA extraction and FecB genotyping

4

Journal Pre-proof Genomic DNA was extracted using the RelaxGene Blood DNA System (TIANGEN, Beijing, China) according to the manufacturer’s protocol. The ratio of absorbance at 260 nm and 280 nm was used to assess the quality of DNA with the Nano-Drop spectrophotometer (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, USA). An OD ratio of 1.7 to 1.9 was accepted as good quality DNA. The integrity of the DNA was assessed using 1.0 % (w/v) agarose gel electrophoresis, and samples showing good DNA quality were selected for further study. The DNA samples were dissolved in TE buffer and stored at -20 °C. The FecB genotyping was carried out by forced PCR-RFLP using primers as described by Wilson et al. [4]. The PCR reaction mixture consisted of 1× PCR buffer, 250 μM dNTP mix, 0.5 μM of each primer, 1.5 U Taq DNA Polymerase (TaKaRa, Dalian, China) and 80 ng of template DNA in 20μl reaction volume. The following PCR conditions were used: denaturation at 94 ℃ for 5min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 62 ℃ for 30 s and extension at 72 ℃ for 30 s, with a final extension at 72 ℃ for 7min. The primer pair amplified a 140 bp region of the BMPR-1B gene. The 5 μl PCR product was digested with 7 U AvaII restriction enzyme (TaKaRa, Dalian, China) at 37 ℃ for 3 h, and run in a 3.0% agarose gel electrophoresis. The FecBBB (homozygous carrier, BB) individual showed 110 and 30 bp bands, FecBB+ (heterozygous carrier, B+) showed 140, 110 and 30 bp bands and the FecB++ animals (non-carrier, ++) revealed an uncut 140 bp band. 2.4 Estrus synchronization and artificial insemination Estrus synchronization was performed in November, within the usual breeding period (August-November) described for this breed in this area. A total of 80 ewes were synchronized with intravaginally inserted sponges (containing 30 mg fluorogestone acetate, Jiahe, Harbin, Heilongjiang, China) for 12 days, and treated with 250 IU of PMSG (Sansheng, Sansheng pharmaceutical Ltd., Ningbo, China) by intramuscular (IM) injection immediately after sponge removal. From 24 h after sponge removal, all ewes were checked regularly (at 12 h intervals) for estrus using an intact ram fitted with an apron. Estrus detection continued until 96 h after sponge removal, when no ewes were mounted by the ram. Cervical insemination was performed on 73 estrous ewes using diluted fresh semen collected 5

Journal Pre-proof from a STH ram of proven fertility. The ejaculate collected from the ram using an artificial vagina was immersed immediately in a warm water bath at 35 ℃ until its assessment in the laboratory. The volume of ejaculate was measured using a micropipette, and sperm concentration was determined by means of a hemocytometer. Semen was diluted (1:10 to 1:15) to a final sperm concentration of 2.5 ×108/ml at 35 ℃ . Cervical insemination with 0.25 ml diluted fresh semen was performed after 12 h from the onset of estrus by using an insemination pipette attached to a syringe with no needle. To achieve satisfactory results, cervical insemination was performed twice at 12 h intervals. Pregnancy diagnosis was performed using a transrectal B-mode ultrasound scanner (EI Medical IBEX LITE, Loveland, USA) at 60 d after cervical insemination. The litter size was recorded at birth and the lamb survival was recorded at weaning on the 60th day. The body weights of lambs were measured at birth and weaning. 2.5 Multiple ovulation and embryo collection Multiple ovulation was performed in August of the following year. The estrous periods of 60 ewes were synchronized with intravaginally inserted sponges (same as used in estrus synchronization) for 12 d, irrespective of the natural estrous cycles. For superovulation, donors were treated with pig FSH (Sansheng, Sansheng pharmaceutical Ltd., Ningbo, China) over a 4 d period, at 12 h intervals, starting 2.5 d before sponge removal. The total dose of FSH for superovulation was 5.5 IU per kilogram of body weight. A total of 0.1 mg cloprostenol (Sansheng, Sansheng pharmaceutical Ltd., Ningbo, China) was administrated at the time of the seventh FSH administration. A total of 100 IU pig LH (Sansheng, Sansheng pharmaceutical Ltd., Ningbo, China) was administrated 48 h after sponge removal to induce ovulation. Eight hours after LH injection, laparoscopic insemination was performed using fresh semen collected from the ram used for AI. All ewes were fasted for 24 h prior to laparoscopic insemination. On day 8 after sponge withdrawal, embryo recovery surgery was performed after ewe anesthesia with sodium pentobarbital. All ewes were fasted for 36 h prior to the surgery. Firstly, the number of functional corpora lutea (CL) was determined by laparoscopy. If ewes had more than 3 CL (considered as a response to treatment), embryos were collected immediately after CL count. Each uterine horn was flushed separately with 40 mL of pre-warmed phosphate-buffered 6

Journal Pre-proof saline supplemented with 1% bovine serum albumin (BSA, A1933, Sigma-Aldrich, St Louis, MO, USA). After embryo recovery, the uterine horns were externally washed with 30 ml of phosphate-buffered saline in order to minimize the development of post-operative fibrous adhesions. The ova/embryos were evaluated using a stereoscopic microscope (Olympus, Tokyo, Japan), and classified based on their stage of development and morphology according to the Manual of the International Embryo Transfer Society (IETS). 2.6 Statistical analysis Statistical analysis was performed with SPSS software, v.18.0 (SPSS Inc. Chicago, IL, USA). Differences in estrus times, number of embryos, fertilization rates, litter sizes, body weights, and survival rates were analyzed using a one-way ANOVA, followed by Duncan's post-hoc test. Normality of the data was assessed using a Shapiro-Wilk test, and homogeneity of variance was verified using a Levene's test. Differences in estrus rates and pregnancy rates were analyzed using a chi-square test procedure. Differences were considered to be statistically significant at P<0.05. Data are presented as means ± S.E.M.

3. Results The distribution of the different genotypes and alleles for the FecB gene are shown in Table 1. The frequencies of BB, B+ and ++ genotypes were 0.5065, 0.4026, and 0.0909, respectively. The allele frequencies of B and + were 0.7078 and 0.2922, respectively. The frequency of the B allele was higher than 0.7. Table 1. The genotype and allele frequencies for FecB in Small Tail Han ewes. The effect of FecB gene is due to a substitution of the 249th amino acid, from glutamine to arginine (Q249R), in the BMPR-1B gene. The overall estrous responses of the different FecB genotype groups are shown in Table 2. During the 96 h observation period after the end of estrus synchronization, more than 90.00% of ewes exhibited overt estrus signs, and no differences were observed among groups (P=0.878). Three ewes from the BB group (10.00%), two ewes from the B+ group (6.67%), and two ewes from the ++ group (10.00%) did not show any overt signs of estrus during the observation period. Estrus onset occurred between 36 and 60 h after sponge removal (P=0.624). The mean intervals 7

Journal Pre-proof from sponge withdrawal to estrus onset were similar among three groups (42.671.61, 44.141.39, 46.671.91 h for BB, B+, and ++, respectively), The duration of estrus was also similar among groups (P=0.829). Table 2. Effect of FecB genotype on estrous performance following estrus synchronization. The pregnancy rates, litter sizes, lambing rates and fecundity rates recorded for the different groups are shown in Figure 1. On day 60 after artificial insemination, there was no difference in pregnancy rate among the groups (P=0.799; Fig. 1A). Compared to ewes in the non-carrier group (++), the litter size of B+ ewes was 0.88 higher, which further increased by 0.41 in BB ewes (P=0.001; Fig. 1B). Meanwhile, the lambing (Fig. 1C) and fecundity (Fig. 1D) rates in B allele carrier groups were higher than those in the non-carrier group (P=0.001). Fig. 1. Effects of FecB genotype on lambing performance following artificial insemination. Number of ewes following artificial insemination: FecBBB genotype group, n=27; FecBB+ genotype group, n=28; FecB++ genotype group, n=18. Different letters indicate a significant difference (P<0.01). Among the 60 donors, 57 responded to FSH superstimulation and was induced ovulation with LH

(more than 3 CL in the two ovaries), with an overall frequency of 95.00%. The functional

CL had good morphological appearance in agreement with their age. The ovarian response and embryo production are shown in Table 3. In the BB, B+, and ++ groups, the proportions of ewes showing a superovulatory response were 95, 100, and 90%, respectively; FecB did not influence the response to multiple ovulation treatment. In terms of CL and recovered embryo numbers, there were no differences among groups (P>0.05). In addition, the fertilization rates were similar (P=0.800) and exceeded 87.00% in all genotype groups. Furthermore, the numbers of grade 1-2 embryos were also similar among groups (P=0.912). Table 3. Effects of FecB genotype on ovarian response and embryo production following multiple ovulation. The body weights and survival rates of lambs born from different FecB genotype groups were shown in Table 4. The FecB genotype significantly affected offspring body weights at birth and weaning. The mean birth (P<0.001) and weaning (P=0.008) weights of lambs born from the BB and B+ groups were lower than those of lambs from the ++ group. There was no difference in the survival rate of lambs at weaning among groups (P=0.327). 8

Journal Pre-proof Table 4. Effects of FecB genotype on body weight and survival rate of the offspring. A, B Superscripts

in different rows in the same column indicate significant differences (P<0.01).

9

Journal Pre-proof 1

4. Discussion

2

STH sheep are popular in China due to their hyper-prolificacy (mean litter size alive=2.61)

3

and year-round estrus [32]. The FecB gene has been identified in STH sheep and has been reported

4

to increase ovulation rate and litter size [7-9, 32]. In this study, the BB and B+ genotypes were

5

dominant in this breed and the frequency of the B allele was 0.71 (shown in Table 1), which is

6

similar to previous reports [9]. These results indicated that the B allele is not completely fixed in

7

STH sheep.

8

Estrus synchronization and AI are important ARTs in sheep production and can help improve

9

reproductive efficiency [33, 34]. In this study, there were no significant differences among the

10

three genotype groups in terms of estrus detection rate, time to estrus onset, and estrus duration

11

within 96 h after sponge withdrawal. The pregnancy rate at 60 d after artificial insemination was

12

similar among the three genotype groups. However, the B allele had an additive effect on litter size

13

after estrus synchronization treatment, and these results are similar with those of previous studies

14

in STH sheep following natural estrus (one copy: increase of 0.22-1.11 lambs; two copies: an

15

additional increase of 0.05-0.97 lambs; based on nine studies) [9]. Our results provide additional

16

evidence that ewes carrying the B allele produce more ova than non-carriers following estrus

17

synchronization. Moreover, the B allele increased lambing and fecundity rates and improved sheep

18

production, which were consistent with other reports under natural reproduction [7, 11, 13] . These

19

results further clarified the fecundity effect of FecB gene [9, 14].

20

MOET is a powerful method for increasing the contributions of good quality ewes and for

21

genetic improvement in sheep. Previous studies have shown that ewes carrying the B allele had a

22

large number of ovulatory follicles which were smaller than those from non-carrier animals [30,

23

35]. Considering the key role of FSH during antral follicle development, a number of studies have

24

been conducted to examine the effect of the B allele on FSH concentration, which have yielded

25

limited and contradictory results. Some studies reported that the concentration of FSH in

26

homozygous carriers was higher than that in noncarrier ewes [36]. Another study, however,

27

reported no differences in the concentration or pattern of FSH secretion between the different

28

genotype groups [30]. Overall, there were no significant differences in the secretion patterns of

29

pituitary gonadotropins or ovarian hormones between the B allele carrier and non-carrier ewes

30

[30-31]. Thus, the higher ovulation rate in ewes carrying the B allele may not be crucially 10

Journal Pre-proof 31

determined by higher concentration of circulating FSH, but may be attributable to the effects of

32

the B allele on follicular development and oocyte ultrastructure at the early stage [16]. In the

33

present study, higher but similar ovulation rates were obtained in all groups as the FSH

34

concentration was increased (administered identical high-doses of exogenous FSH). Similarly,

35

Hudson et al. [36] reported that the ovulation rates in homozygous carriers and non-carriers given

36

an identical plasma concentration of FSH were not significantly different. The fertilization rates

37

were also similar among groups, and all exceeded 87.00%. Furthermore, there were no significant

38

differences in the numbers of grade 1-2 embryos among groups. Overall, our preliminary results

39

indicate that the presence of the B allele did not improve the yield and quality of embryos after

40

multiple ovulation, nor did it significantly influence ovarian response to exogenous FSH

41

superstimulation.

42

Our results indicated that the B allele had a negative effect on early postnatal body weight of

43

offspring. The birth and weaning weights of lambs born from BB and B+ ewes were

lower than

44

those of lambs born from ++ ewes (P<0.01) due to the high fecundity. These results were similar

45

to those of previous studies [12, 37, 38]. Litter size is an important factor affecting lamb survival

46

especially under extensive production systems. In this study, the higher litter sizes of BB and B+

47

groups were associated with a slight reduction in lamb survival before weaning, although the

48

difference was not statistically significant. Most lamb losses occur before weaning, and only about

49

10% of lamb deaths occur after weaning [39]. Therefore, mortality after weaning was not

50

considered in this study. Similar trends of survivability have been also reported previously [37, 40].

51

Overall, the reproductive advantage of ewes carrying the B allele was partly counteracted by

52

poorer growth of their lambs.

53

5. Conclusions

54

In the present study, the frequency of the B allele of the FecB (BMPR-1B mutation) gene was

55

higher than 0.7, and the BB and B+ genotypes were dominant in STH sheep. The presence of the B

56

allele does not appear to have a significant influence on parameters of estrus synchronization in

57

ewes. After cervical artificial insemination, the pregnancy rates at 60 d were similar among the

58

three genotype groups. The B allele had an additive effect on litter size following estrus

59

synchronization treatment, and these results are similar to those of previous studies in this breed 11

Journal Pre-proof 60

after natural estrus. The presence of the B allele did not influence the ovarian response to

61

high-dose FSH stimulation, nor improve yield or quality of embryos after multiple ovulation.

62

Furthermore, the higher prolificacy in the ewes carrying the B allele was associated with a

63

reduction in the body weight of offspring at birth and weaning.

64

Acknowledgments The present work was supported by the Natural Science Foundation of Heilongjiang Province

65 66

of

China

(C2017026).

12

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18

Journal Pre-proof Revised Highlighted The presence of B allele does not appear to have a significant influence on parameters of estrus synchronization in ewes. The B allele had an additive effect on litter size after estrus synchronization treatment. The presence of B allele did not influence the ovarian response to high-dose FSH stimulation, nor improve yield or quality of embryo after multiple ovulation. The higher prolificacy in the ewes carrying the B allele was associated with a reduction in offspring body weight at birth and weaning.

Journal Pre-proof Table 1. The genotype and allele frequencies for FecB in the Small Tail Han ewes. The effect of FecB gene is due to a substitution of the 249th amino acid from glutamine to arginine (Q249R) in the BMPR-1B gene.

Genotype frequency

Allele frequency

Gene

Number of ewes (n)

BB

B+

++

B

+

FecB gene (BMPR-IB mutation)

231

0.51

0.40

0.09

0.71

0.29

Journal Pre-proof 1

Table 2. Effect of FecB genotype on estrous performance following estrus synchronization. Estrus onset after sponge removal (n)

Number of ewes (n)

24h

36h

48h

60h

≥72h

No estrus signs

FecBBB

30

1

12

12

2

0

FecBB+

30

0

11

15

2

FecB++

20

0

5

10

3

Genotype

Time to estrus onset (h)

Ewes in estrus (%)

Estrus duration (h)

3

42.671.61

90.00% (27/30)

23.111.91

0

2

44.141.39

93.33% (28/30)

22.291.60

0

2

46.671.91

90.00% (18/20)

21.332.48

Journal Pre-proof 3

Table 3. Effects of FecB genotype on ovarian response and embryo production following

4

multiple ovulation.

Genotype

Ewe donors (n)

FecBBB

20

FecBB+

20

FecB++

20

Response to treatment (%) 95.00 (19/20) 100.00 (20/20) 90.00 (18/20)

Average corpora lutea count (n)

Total embryos recovered (n)

Fertilization rate (%)

Number of grades 1-2 embryos (n)

14.260.95

9.160.79

87.874.15

7.110.80

12.351.01

8.200.77

91.763.35

6.700.73

13.110.68

8.440.61

88.895.46

7.060.67

Journal Pre-proof 6

Table 4. Effects of FecB genotype on body weight and survival rate of the offspring.

7

Superscripts in different rows in the same column indicate significant differences (P < 0.01).

8

Genotype

Number of lambs born (n)

Birth weight (kg)

Survival rates of lambs at weaning (%)

Weaning weight (kg)

FecBBB

46

2.940.096A

87.355.08

13.130.32A

FecBB+

46

2.980.080A

90.054.07

13.850.33A

FecB++

17

3.650.180B

97.252.75

15.120.57B

A, B