Research Article

Association of Growth Differentiation Factor 9 (GDF9) Gene with Birth Type, In vitro Maturation and Selected Physiological Traits in Awassi Sheep

Hadi Awad Hassooni1, Safaa Sabbar Atiyah2*, Alaa S. J. Mohamed1

1University of Al-Muthanna, Faculty of Agriculture, Department of Animal Production, Al-Muthanna, Iraq; 2Al-Furat Al-Awsat Technical University, Technical Institute of Kufa, Department of Animal Production Techniques, Kufa, Iraq.

Received | December 17, 2024; Accepted | May 26, 2025; Published | July 03, 2025

*Correspondence | Safaa Sabbar Atiyah, Al-Furat Al-Awsat Technical University, Technical Institute of Kufa, Department of Animal Production Techniques, Kufa, Iraq; Email: [email protected]

Citation | Hassooni HA, Atiyah SS, Mohamed ASJ (2025). Association of growth differentiation factor 9 (GDF9) gene with birth type, In vitro maturation and selected physiological traits in awassi sheep. J. Anim. Health Prod. 13(3): 617-623.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.3.617.623

ISSN (Online) | 2308-2801

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Awassi sheep are the most widespread breed in Iraq and represent more than half of the local sheep breeds. Selecting genetic variety and key genes for genetic improvement is made possible by incredible developments in statistics and molecular biology. Selecting individuals with the finest phenotypic appearance is the basis of traditional genetic improvement of farm animals. In the area of genetic manipulation, it has also attained good commercial viability. Since economic features are influenced by the dominance of several genetic sites, sometimes referred to as quantitative trait sites, scientific advancements have also contributed to a better knowledge of how the genome functions, allowing for the creation of more precise selection programs with less effort and expense. by choosing these linked sites (Al-Dabbagh, 2019).

One of the most crucial economic characteristics is litter size, which has a direct bearing on the economic benefits from contemporary sheep husbandry. In order to improve litter size using marker-assisted breeding, it can be helpful to investigate the main genes influencing litter size. The GDF9 gene, which encodes a member of the transforming growth factor-β (TGF-β) family (Al-Mutar and Younis, 2020), has important functions in ewe reproduction (Vage et al., 2013). The gene is located on chromosome 5 of sheep (Tang et al., 2018) and has two exons separated by an intron of 1126 bases. GDF9 is mostly expressed in oocytes and is crucial for sheep follicle growth and ovulation. (Mullen and Hanrahan, 2014). In previous studies, several mutations in the gene were identified in Belclare sheep in Europe (Eghbalsaied et al., 2012), Afshari sheep (G2, G3, and G4) (Abdoli et al., 2018), fat tailed sheep (G1, G2, G3, and G4) (Saleh et al., 2020), and FecG H in Rahmani sheep in Africa (Bravo et al., 2016). It was reported that c.978A>G, c.994G>A, FecG H, and c.1040T>C (Phe 347 Ser) were associated with birth size in Creole Araucana, Rahmani, and Mongolian sheep, respectively (Tong et al., 2020). The present study aims to clarify the association between GDF9 gene polymorphisms and type of parturition, as well as their relationship with in vitro maturation of Awassi sheep oocytes.

MATERIALS AND METHODS

The experimental design of the current study was in in line with the international animal ethical protocols. Blood samples were collected from ewes immediately before slaughter. Blood samples were collected from the jugular vein using sterile 10 ml medical syringes after thoroughly cleaning the puncture site. A total of 8 ml of blood was collected from each animal. The samples were then divided into two portions. The first portion, comprising 3 ml, was placed into tubes containing an anticoagulant and stored in a refrigerator until DNA extraction was carried out. The second portion, consisting of 5 ml, was transferred into gel tubes without anticoagulant to allow clotting and facilitate serum separation. These tubes were centrifuged at 3000 rpm for 10 minutes. The resulting serum was carefully extracted using sterile syringes, transferred to new sterile test tubes, and stored at –15 °C for subsequent hormone analysis.

Collection of Oocytes

Oocytes were extracted from the ovaries by aspiration, which involved removing the follicular fluid from the large and medium-sized follicles on the ovary’s surface using an 18 mm medical syringe, Since the diameter of the needle influences the flow of sheep oocytes during aspiration and a medical syringe filled with 0.5 ml of RBMI-1640 culture medium and 20 IU/ml of heparin, an anticoagulant, to stop the oocytes from adhering to one another. Following extraction, the oocytes were placed in a Petri plate, and a manually adapted Pasteur pipette was used to collect the oocytes under a dissecting microscope. The Petri dish was used to wash the oocytes three times with culture medium to eliminate the remaining cell components affixed to the oocytes, and the oocytes with a atretic and irregular shape, which were characterized by shrinkage and a wide gap between them and the Zp region, or damage to this region were removed.

Oocyte Classification

After isolating the oocytes in a Petri dish and washing them three times in RBMI-1640 culture medium, the oocytes were classified based on their external appearance according to the method of Nogueira et al. (2009), in terms of whether they contained layers of germinal aggregate cells or not and whether they were mature or immature, as mature, atretic, and abnormally shaped ones were excluded.

In vitro Maturation of Oocytes

After washing the oocytes three times with RBMI-1640 culture medium supplemented with some hormones (5 IU/mL ECG, 10 IU/mL hCG, and 1µg/ml Estrogen) for the purpose of using them as a medium for oocytes in vitro maturation, The oocytes were moved to a four-well petri plate, which was separated into five halves. and 6-4 oocytes were placed equally in each dish containing The usual culture medium, RBMI-1640, was coated with paraffin oil and kept in a CO2% incubator for 24 hours at 38.5°C and 95% relative humidity. To differentiate between mature and immature oocytes, the oocytes were subsequently examined under a microscope.

Statistical Analysis

The data of the current study were analyzed using statistical analysis system (SAS Institute Inc.2018) and analysis of variance (ANOVA). For the analysis, a significance level of p<0.05 was used. The Duncan (1955) multinomial test was used to compare the significant differences between the means using the least squares means approach. Mathematical model of the relationship of genetic conformations of the GDF9 gene to the studied traitswas:

Yil=µ + Gi + eil

RESULTS AND DISCUSSION

The growth differentiation factor 9 (GDF9) gene is an important gene that affects reproductive traits and is also called FecG (Margawati et al., 2023). The growth differentiation factor gene is a member of the large family of transforming growth factors (TGF-β) (Al-Mutar and Younis, 2020; Elshareef et al., 2023; Hassooni et al., 2024). GDF9 is mainly expressed in oocytes and plays an important role in follicle development and ovulation in sheep. It also has important functions in ewe reproduction, including its favorable effect on ovulation rate and reproduction (Tang et al., 2018; Munoz et al., 2021). Several in vitro studies were conducted to determine the effect of the genotypes of the GDF-9 gene on the growth and vitality of ovarian follicles. Mery et al. (2007) took 2348 follicles at different stages of growth from the ovaries of five ewes and cultured them in the laboratory. They found that the speed of development of these follicles was linked to the gene expression of the GDF-9 gene and its receptors.

Electrophoresis was performed using 1% agarose gel as described in the Materials and Methods section. Each sample consisted of 5 µL of DNA mixed with 2 µL of loading dye, and was loaded into the wells of a 1% agarose gel prepared with Diamond™ Nucleic Acid Dye (Promega, USA). The dye was diluted in TE buffer at a ratio of 1:100 (v/v) using a micropipette and applied to the gel. After setting the appropriate voltage, DNA migration was visualized using a UV gel documentation system, and images were captured using a dedicated gel imaging camera (Figure 1).

 

Relationship Between Genetic Polymorphisms in the Studied Segment of the GDF9 Gene and the Percentages of Cumulus Cells in Awassi Sheep Oocytes

Animals carrying the three genetic variants (GG, GA, and AA) resulting from the mutation at position 205 of the examined segment of the GDF9 gene did not show significant differences (P > 0.05), as presented in Table 1. This finding applies to in vitro maturation outcomes of mature, immature, and atretic oocytes in the presence of cumulus cells, taking into account the type of birth (single or twin).

 

Table 1: The relationship between the genetic polymorphisms of GDF9 gene (G<A) and in vitro maturation rates of Awassi sheep oocytes with cumulus cells (mean ± standard error).

Genetic polymorphisms	Type of birth	Presence of cumulus cells
GG	Single	Atretic oocytes	Immature oocytes	Mature oocytes
	Twins	9.63±2.10	14.21±2.41	76.14±4.28
GA	Single	13.20±4.08	13.48±3.83	73.29±7.67
	Twins	14.16±2.26	20.76±3.77	65.06±5.72
AA	Single	18.87±8.06	20.25±5.23	60.87±11.5
	Twins	10.68±2.52	18.01±5.42	71.31±7.41
Significance level		P>0.05	P>0.05	P>0.05

 

In cases of single births, the percentages of atretic oocytes were 9.63% for GG, 14.16% for GA, and 10.68% for AA genotypes. The corresponding values for immature oocytes were 14.21%, 20.76%, and 18.01%, while mature oocytes showed percentages of 76.14%, 65.06%, and 71.31% for GG, GA, and AA, respectively. For twin births, atretic oocyte percentages were 13.20% (GG), 18.87% (GA), and 16.27% (AA). Immature oocyte percentages were 13.48%, 20.25%, and 18.77%, and mature oocytes were 73.29%, 60.87%, and 64.94% for GG, GA, and AA genotypes, respectively.

Although these values showed some variation, they were not statistically significant, suggesting that the GDF9 polymorphism at site 205 may not have a notable effect on oocyte maturation outcomes when cumulus cells are present. These findings differ from those reported by Abdelgadir et al. (2021) in Sudanese sheep, possibly due to breed-specific genetic differences affecting oocyte development and maturation. This highlights the importance of considering genetic background in reproductive biotechnology studies.

Association Between Polymorphisms in the Studied Segment of the GDF9 Gene and the Absence of Cumulus Cells in Awassi Sheep Oocytes

The results presented in Table 2 showed significant differences (P ≤ 0.05) among genetic variants in the in vitro maturation of immature oocytes from Awassi sheep in the absence of cumulus cells. However, no significant differences were observed in mature and atretic oocytes, regardless of whether the birth type was single or twin. The GA genotype showed superior performance in twin births for immature oocytes.

In the case of single births, the percentages for atretic oocytes were 21.37% (GG), 27.53% (GA), and 25.86% (AA); for immature oocytes, 34.55% (GG), 25.08% (GA), and 38.91% (AA); and for mature oocytes, 39.63% (GG), 47.38% (GA), and 46.68% (AA).

 

Table 2: The relationship between the genetic polymorphisms of GDF9 gene (G<A) and in vitro maturation rates of Awassi sheep oocytes without cumulus cells (mean ± standard error).

Genetic polymorphisms	Type of birth	Absence of cumulus cells
GG	Single	Atretic oocytes	Immature oocytes	Mature oocytes
	Twins	9.63±2.10	14.21±2.41	76.14±4.28
GA	Single	13.20±4.08	13.48±3.83	73.29±7.67
	Twins	14.16±2.26	20.76±3.77	65.06±5.72
AA	Single	18.87±8.06	20.25±5.23	60.87±11.5
	Twins	10.68±2.52	18.01±5.42	71.31±7.41
Significance level		P>0.05	P≤0.05	P>0.05

 

For twin births, the percentages for atretic oocytes were 23.85% (GG), 21.85% (GA), and 21.82% (AA); for immature oocytes, 29.42% (GG), 45.01% (GA), and 38.91% (AA); and for mature oocytes, 46.71% (GG), 33.75% (GA), and 39.26% (AA).

 

The observed variations in these results may be attributed to differences in genetic composition, the number of animals used, and the average age of the sheep, as well as environmental conditions across experimental settings.

 

Relationship Between Genetic Variants of the Studied GDF9 Gene Segment and LH and FSH Hormone Concentrations in Single- and Twin-Born Awassi Sheep

The hormonal analysis presented in Figure 2 revealed no significant effect (P ≥ 0.05) of the genetic variants (GG, GA, and AA) resulting from the mutation at position 205 of the GDF9 gene segment on the concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in twin-birth Awassi sheep. The observed hormone levels (in ng/ml) for LH were 0.36 (GG), 0.30 (AA), and 0.28 (GA), while FSH concentrations were 0.41 (GG), 0.34 (AA), and 0.36 (GA), respectively. A slight decrease in hormone levels may have resulted from animal stress during blood collection, as previously reported by Kohanski and Redmond (2017).

 

Similarly, Figure 3 shows no significant differences (P ≥ 0.05) in LH and FSH concentrations across genotypes in single-born Awassi sheep. LH levels were 0.28, 0.34, and 0.28 ng/ml for the GG, GA, and AA genotypes, respectively. FSH levels were recorded at 0.33, 0.35, and 0.54 ng/ml for the same genotypes. These results are not consistent with the findings of Jumaa and Kassim (2022), who reported that hormonal treatments significantly affected reproductive organ development and sex hormone concentrations. The discrepancies may be due to differences in the physiological, morphological, and structural characteristics of the animals used, particularly in ovarian structure and hormone production. Additionally, environmental factors and management conditions may have influenced the hormonal activity and reproductive performance in the studied sheep (Ahmed and Atiyah, 2022).

Reproductive traits in ewes are strongly influenced by the GDF9 gene, a member of the TGF-β (Transforming Growth Factor Beta) superfamily. GDF9 is predominantly expressed in oocytes and plays a critical role in follicular development and ovulation (Tang et al., 2018). It has been identified as one of the key genes contributing to sheep prolificacy (Vage et al., 2013; Mullen and Hanrahan, 2014; Souza et al., 2014), by enhancing oocyte developmental competence and regulating ovarian function (Stocker et al., 2020).

Moreover, GDF9 interacts with other key factors in the TGF-β signaling pathway, including BMP15 and BMPR1B. These molecules work together to control oocyte maturation, follicular atresia, and granulosa cell differentiation (Liu et al., 2019). GDF9 and BMP15 are secreted by oocytes and bind to BMPR1B receptors on granulosa cells, thus potentially increasing litter size and ovulation rate. The combined action of GDF9 and BMP15 also promotes the expression of anti-Müllerian hormone (AMH), which is critical for ovarian function and follicular recruitment (Roy et al., 2018). Previous studies have shown that sheep with concurrent mutations in FecB and BMP15 had significantly higher litter sizes than those with only one mutation (Chu et al., 2007). Similarly, Hanrahan et al. (2004) demonstrated that sheep carrying both GDF9 and BMP15 mutations ovulate more frequently than those with only one mutation. However, the interaction between BMPR1B and GDF9 remains poorly understood and warrants further investigation.

Additionally, the present study observed a significant superiority in in vitro maturation (IVM) rates of oocytes containing cumulus-oocyte complexes (COCs) compared to those without cumulus cells. This may be due to the crucial physiological role of the cumulus cell layer, which supports both cytoplasmic and nuclear maturation of the oocyte by maintaining it in the germinal vesicle (GV) stage and facilitating molecular exchange between the oocyte and its microenvironment (Shimada and Terada, 2002).

In contrast, the current findings disagree with Singh et al (2020), possibly due to several factors affecting oocyte quality. These include animal age, season of ovary collection, time elapsed between slaughter and oocyte retrieval, environmental conditions during collection, nutritional status, and the functional state of the ovary at the time of sampling (Amer et al., 2008). These variables can significantly impact oocyte competence and maturation success in vitro.

CONCLUSIONS AND RECOMMENDATIONS

The current study suggests that the GDF9 gene and litter size in Awassi ewes could serve as biological indicators of ovulation and potentially be used to enhance fertility. Mutations in the GDF9 gene represent important genetic resources for livestock production. These mutations influence growth factor profiles, which, in turn, affect litter size. Further investigation is needed to understand how GDF9 interactions impact follicular growth. Since abnormal expression of GDF9 can lead to infertility, breeders should recognize the importance of this gene as a marker for fertility.

ACKNOWLEDGEMENTS

The administration and staff of the Al-Nahrain Station in Al-Qadisiyah are acknowledged by the authors for their help in completing the study’s initial phase. A special thanks goes out to the Animal Production Department and Postgraduate Studies Laboratory at Al-Muthanna University’s College of Agriculture, which provided all the resources required to complete the second half of his research.

NOVELTY STATEMENTS

This work aims to provide us a better knowledge of the connection between significant reproductive and physiological features in Awassi sheep and genetic polymorphisms in the growth and differentiation factor 9 (GDF9) gene. Since the majority of earlier studies on the GDF9 gene and how it affects fertility have concentrated on sheep breeds from around the world. Awassi sheep are a significant native breed with distinctive traits in our area, particularly in Iraq and the Middle East. Information that might not be available in other breeds is revealed by specifically examining the GDF9 gene in the Awassi breed. The importance of GDF9 in sheep fertility has found specific correlations between GDF9 genotypes, parturition type (number of litters), in vitro maturation success, and selected physiological traits. Other physiological traits include lamb birth weight, growth rate, and even the length of the breeding season in Awassi sheep. This information is important for developing breeding improvement techniques and artificial insemination. This information not only fills a research gap but also provides a strong scientific foundation for developing targeted genetic improvement strategies to boost fertility and productivity in the Awassi breed.

AUTHOR’S CONTRIBUTIONS

The design of the experiment, in vitro oocyte maturation (IVM) phase, samples collection and molecular biology tools to analyse genetic diversity was contributed by Safaa S. Atiyah and Hadi A. Hassooni. As the field supervisor, Hadi A. Hassooni oversaw the gathering of information from Awassi sheep flocks on physiological characteristics and parturition type. He also oversaw the statistical analysis of the data to connect these characteristics to the genotypes that were found. Alaa S. J. Mohamed was supported both theoretical and practical elements, reviewing scientific literature, interpreting results, and highlighting the study’s importance.

Conflict of Interest

Regarding the current work, the authors state that they have no conflicts of interest.

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