Reproduction of Sheep through Nuclear Transfer of Somatic Cells: A Bibliometric Approach

Simple Summary Different reproductive biotechnologies have been applied to sheep, such as cloning, which has been successfully applied in this species. In this context, the aim of the present study was to carry out a bibliometric analysis of the scientific literature on cloning applied to sheep reproduction, since the first report was published, to identify the most cited articles, main authors and collaboration among them, published journals, institutions with more published papers, most prolific countries and the network collaboration among them, and research topics. This study collected bibliographic data from 124 papers relating to cloning of sheep. The articles that were cited more often addressed topics related to the generation of transgenic animals, recovery of wild species, and xenotransplants. So far, no bibliometric studies have been conducted about cloning of sheep. Abstract Somatic cell nuclear transfer (SCNT) is a reproductive biotechnology with great potential in the reproduction of different species of zootechnical interest, including sheep. This study aimed to carry out a bibliometric analysis of scientific papers published on the application of SCNT in sheep reproduction during the period 1997–2023. The search involved the Science Citation Index Expanded and Social Sciences Citation Index databases of the main collection of the Web of Sciences with different descriptors. A total of 124 scientific papers were analyzed for different bibliometric indicators using the VOSviewer software. Since 2001, the number of SCNT-related papers that have been published concerning sheep reproduction has increased and it has fluctuated in ensuing years. The main authors, research groups, institutions, countries, papers, and journals with the highest number of papers related to the application of SCNT in sheep reproduction were identified, as well as the topics that address the research papers according to the terms: somatic cell, embryo, oocyte, gene expression, SCNT, and sheep.


Introduction
Sheep are one of the first species to have been domesticated from about 8000 to 9000 years ago. The adaptability of this species to different types of weather has allowed their wide geographic distribution. The global population of sheep reached 1.2 billion by the year 2012. Sheep are among the top five most economically important domestic species in the world, and approximately 1400 discrete breeds have been registered [1]. Sheep are easy to manage and maintain, and their pregnancy period is relatively short, which are advantageous for evaluating genetic improvement programs [2,3].
There are different reproductive biotechnologies that have focused on sheep which include artificial insemination (AI), in vitro fertilization (IVF), gamete and embryo cryopreservation, cloning, among others [4]. In vitro production of cloned embryos involves vertical and horizontal approaches [5]. The vertical approach includes the generation of monozygotic twins from blastomere separation and embryo bipartition in vivo or by IVF [6]. In contrast, the horizontal approach involves somatic cell nuclear transfer (SCNT). In SCNT, an oocyte devoid of its nucleus serves as a cytoplasmic receptor for donated genetic information from a somatic cell [7]. The first attempts to generate cloned embryos by blastomere separation and embryo bipartition were carried out in sheep, as was the generation of embryos by SCNT [8]. SCNT is a biotechnology with great potential to reproduce sheep with high genetic value [9], to conserve endangered wild sheep [10], and to generate transgenic sheep with biomedical purposes [11].
The scientific and technological advances in the past several decades have been reflected in an increase in scientific information in bibliographic databases for the dissemination of knowledge. This has enhanced the use of bibliometrics [12]. Bibliometrics is a method to help evaluate scientific information by evaluating a set of methodological knowledge in published papers through indicators, number of papers published, and citations of these papers, according to the region or country of origin, authors, working groups, and research centers [13]. Bibliometric studies have been used to quantify scientific output and to identify groups and areas of excellence, thematic and interdisciplinary emerging disciplines, and thematic collaboration networks [12]. Governments can use this information to implement policies that benefit the scientific and technological development of their nations [14].
An evaluation of the different elements of scientific papers can reveal different bibliometric indicators that measure the results of scientific and technological work. The choice of the database to be used in the analysis of scientific information will condition the bibliometric indicators that can be developed [12].
The aim of this study was to identify regularities of scientific information to provide an overview of scientific research published in mainstream journals on the application of SCNT in sheep reproduction. The study used several one-dimensional and multi-dimensional bibliometric indicators. The data were analyzed using the VOSviewer software.

Materials and Methods
The Science Citation Index Expanded (SCIE) and the Social Sciences Citation Index (SSCI) databases of the Web of Science were used to search for papers related to the application of SCNT in sheep reproduction that had been indexed in these databases and published in mainstream journals [15] from 1997 to 2023. The expressions used in the advanced search option in the search performed in January 2023 were TS = ("Somatic cell*" "Nuclear transfer") OR SCNT) and TI = (Ewe OR Sheep OR Ovine OR "Lamb*"). These words were searched for in the titles, abstracts, and keywords of the scientific papers. Only research and review papers were considered. The bibliographic records that were obtained were analyzed according to the one-dimensional and multi-dimensional indicators [16,17] shown in Table 1. Table 1. Bibliometric indicators used to analyze scientific research on the application of SCNT in the production of sheep indexed in the Web of Science. The bibliographic data were analyzed using Excel to obtain the "literature growth" indicator. Data obtained from the other indicators were analyzed using the VOSviewer software version 1.6.19 (Centre for Science and Technology Studies, Leiden University, Leiden, The Netherlands, 2023), which visualizes scientific landscapes. This bibliometric software was used, in the present study, to create and visualize maps by "visualization of similarities" (VOS), a method proposed by van Eck and Waltman [18]. This is an alternative to multi-dimensional scaling to visualize similarities between themes or objects. A cooccurrence analysis of the words related to sheep SCNT was performed in the titles and abstracts of the scientific papers [19]. The generated maps of science featured scientific thematic networks between teams of researchers, institutions, and countries concerning the application of SCNT in sheep. The terms were standardized before analysis. A joint word analysis was also performed, which was extracted from the titles and abstracts of the published papers. This co-occurrence of words reflects the conceptual relationship network of the views of scientists active in the field. The frequency of words was used to construct co-occurrence maps representing the intellectual content of an area of research through the analysis of groups and networks [20].

Growth of Literature
The Science Web reference and citation database revealed 107 papers. In addition, 17 papers were included from the Journal Citation Reports (JCR) that had not been identified in the initial search. In total, there were 124 papers that comprised 118 research papers (95.2%) and 6 review papers (4.8%). The first paper on the application of SCNT in sheep, entitled "Viable offspring derived from fetal and adult mammalian cells", was published in 1997 in the journal Nature [21]. In subsequent years from 2001, the number of published papers fluctuated from one to twelve papers every year, with an annual average of five papers. Most papers (n = 12) were published in 2013 ( Figure 1).

Most Productive Author
Keith Henry Stockman Campbell published the most papers about SCNT in sheep. The 15 articles published have been cited 4868 times (H-index of 34) [22]. Dr. Campbell was affiliated with the University of Nottingham until his death in 2012. One aspect of his research was the application of SCNT in mammals, using sheep as a research model for the generation of cloned or transgenic lambs. The author with the highest scientific productivity is Professor Sir Ian Wilmut, who has published nine articles that have been cited 4992 times (H-index of 69). His research at the University of Edinburgh focused on cell reprogramming mechanisms and regenerative medicine ( Table 2). papers were included from the Journal Citation Reports (JCR) that had not been identified in the initial search. In total, there were 124 papers that comprised 118 research papers (95.2%) and 6 review papers (4.8%). The first paper on the application of SCNT in sheep, entitled "Viable offspring derived from fetal and adult mammalian cells", was published in 1997 in the journal Nature [21]. In subsequent years from 2001, the number of published papers fluctuated from one to twelve papers every year, with an annual average of five papers. Most papers (n = 12) were published in 2013 ( Figure 1).

Cooperation between Authors
There are five research groups that focused on the application of SCNT in sheep (

Published Papers and Cooperation among Countries
The countries that have most intensively performed and published research on SCNT involving sheep are shown in Table 3

Published Papers and Cooperation among Countries
The countries that have most intensively performed and published research on SCNT involving sheep are shown in Table 3. The collaboration among countries is shown in Figure 3. Scholars from China have collaborated mainly with colleagues from Iran, Australia, Canada, and United States. Scholars from England have collaborated with colleagues from Scotland and France. Finally, academics from Italy have collaborated with colleagues from Poland. Table 3. Main countries with the most published papers concerning the application of SCNT in sheep production.

Institutions with the Largest Contribution of Published Papers
The University of Nottingham has the highest number of published papers concerning the application of SCNT in sheep, with 14 papers. In accordance with the Academic Ranking of World Universities (ARWU) and the World Classification of Universities (QS), it is ranked 101-150th and 114th, respectively. The Roslin Institute of the University of Edinburgh holds the second position, with 13 published papers that have been cited 1844 times, seven papers have been cited more than 100 times each. The University of Edinburgh occupies position 35th in the ARWU and position 15 in the QS (Table 4).

Journals
The eight main journals with papers dealing with SCNT-related topics in sheep are shown in Table 5. The information in the table includes bibliometric data and impact factor (IF) [23]. It also includes the positions of the journals in accordance with the thematic category, which indicates the quartile according to the JCR. IF is used as an indicator of the relative importance of a journal within a particular area of study and it evaluates the frequency with which journal articles are cited during a given period [24]. The journal Cellular Reprogramming has published the most papers related to SCNT in sheep (n = 11, IF

Institutions with the Largest Contribution of Published Papers
The University of Nottingham has the highest number of published papers concerning the application of SCNT in sheep, with 14 papers. In accordance with the Academic Ranking of World Universities (ARWU) and the World Classification of Universities (QS), it is ranked 101-150th and 114th, respectively. The Roslin Institute of the University of Edinburgh holds the second position, with 13 published papers that have been cited 1844 times, seven papers have been cited more than 100 times each. The University of Edinburgh occupies position 35th in the ARWU and position 15 in the QS (Table 4).

Journals
The eight main journals with papers dealing with SCNT-related topics in sheep are shown in Table 5. The information in the table includes bibliometric data and impact factor (IF) [23]. It also includes the positions of the journals in accordance with the thematic category, which indicates the quartile according to the JCR. IF is used as an indicator of the relative importance of a journal within a particular area of study and it evaluates the frequency with which journal articles are cited during a given period [24]. The journal Cellular Reprogramming has published the most papers related to SCNT in sheep (n = 11, IF 2.257, JCR position in the fourth quartile of thematic categories). The next journal is Theriogenology (n = 10, IF 2.923, first quartile for the "Veterinary Sciences" category). The journal with the highest number of citations is Biology of Reproduction (second quartile of the "Reproductive Biology" category), followed by Theriogenology (first quartile in the "Veterinary Sciences" category).

Most Cited Articles
The citation structure of papers between 1997 and 2022 included five papers with ≥180 citations ( Table 6). The most cited article referred to the first lamb cloned from adult cells obtained from mammary gland; this success demonstrated two facts, i.e., first, terminally differentiated cells preserve all their genetic information, and it can be restored; and second, the SCNT can be applied to superior vertebrates such as mammals [21].
The second most cited article focused on the generation of transgenic lambs from the transfection of fetal fibroblasts with the neomycin resistance marker gene and human coagulation factor IX, for protein coding in sheep's milk [25]. This was followed by an article related to the generation of transgenic lambs. It focused on "gene targeting" in fetal fibroblasts to integrate a therapeutic transgene into the gene locus ((COL1A1) collagen type I, alpha 1 chain) in sheep [26]. The fourth most cited article was related to SCNT as a tool for the recovery of wild species at risk, in which the feasibility of using Ovis aries oocytes as receptors of O. orientalis musimon fibroblasts for interspecific SCNT. One of the embryos reached the term of gestation with the birth of a European mouflon [10]. Six other articles were cited from 50 to 99 times. One study [32] evaluated the imprinting status of insulin-like growth factor 2 and H19 gene (IGF2-H19) and insulin-like growth factor 2 receptor (IGF2R) in lambs generated by SCNT. The authors observed that one lamb displayed deregulation in the imprint of the second loci intron of the IGF2R gene.
Three articles were cited from 40 to 49 times. One study [37] evaluated the expression of mitochondrial DNA replication factors encoded by the nucleus and expressed for the first time in the later stages of early embryonic development.
The nine articles with from 30 to 39 citations include Peura and Vajta [39] who described a new SCNT method for cattle and sheep (handmade cloning), characterized by using pelucide-zone-free oocytes and the absence of micromanipulators to enucleate them.
Seven articles were cited from 20 to 29 times. They included Zhang et al. [11] who described generating transgenic lambs with high levels of omega-3 fatty acids using handmade cloning. The authors concluded that handmade cloning efficiency was similar to the conventional technique for the generation of transgenic animals.
Twenty-six articles were cited between 19 and 10 times. Among them, Wen et al. [40] tested two inhibitors of histone deacetylases, trichostatin A, and scriptaid. The latter improved the epigenetic status of ovine embryos via SCNT.
Finally, 52 articles were cited ≤9 times. Choi et al. [41] combined calcium ionophore, strontium chloride, and cytochalasin B to activate cario-cytoplast complexes, improving embryo quality. Among these articles, 10 articles had no record of citations. The most recent article was by McLean et al. [42] who studied the effect of embryo aggregation during the vitrification process of cloned sheep embryos. They observed that the aggregated embryos had an in vivo survival rate similar to that of the group that was not vitrified. The oldest most cited published paper is that of Wilmut et al. [21]. Table 6 includes the articles with the highest number of citations between 1997-2022.

Identification of Research Topics
The results of the co-occurrence analysis revealed 553 words or terms that were used most frequently in scientific papers. Only those with more than five co-occurrences were considered. The resulting 52 terms were organized into five clusters. The 14 words or terms most frequently used in the published papers are listed in Table 7. This analysis clarified the main topics of interest in the study area.  Figure 4 displays five clusters of words (a cluster is a group of words related to each other) and the relationships among the clusters. Each cluster is represented by one color, cluster one has 14 words (red), cluster two includes 13 words (bright green), cluster three involves 9 words (blue), cluster four contains 9 words (light green), and cluster five has five words (purple).

Discussion
In the present study, 124 papers on the reproduction of sheep by applying SCNT were counted in the WOS database; this quantity of papers was similar to that reported in a review carried out for the 25th anniversary of cloning using SCNT. This review published a survey of all published papers (1997-2020) on SCNT classified by species, among which mice, cattle, pigs, goats, and sheep were the species for which more than 100 published papers had been registered [43].
Since the publication of the study on the birth of the Dolly sheep (1997), the number of papers published per year has fluctuated, with an average of five papers per year. However, a more detailed analysis shows that, during the first (1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006) and second (2007-2016) decades, the numbers of published papers were 25 and 72, respectively, while in the last period (2017-2022), 27 papers were published, indicating a general growth in research on the reproduction of sheep by cloning.
China, Italy, England, and Scotland are the countries that have generated more than 50% of the scientific research on the reproduction of sheep using SCNT. China is the world's leading producer of sheep, while Australia, Iran, and the United Kingdom (which includes England and Scotland) are among the ten countries with the highest number of sheep worldwide, according to FAO data for 2020 [44]. This could partly explain the interest of these countries in research on the reproduction of sheep by cloning.
The universities that have shown interest in the reproduction of sheep by cloning, are the University of Nottingham (England), the Roslin Institute of the University of Edinburgh (Scotland), the University of Teramo (Italy), and the China Agricultural University (China), which are the affiliations from the most cited authors in this area of research.
The principal journals that have published topics related to sheep reproduction by applying SCNT are Cellular Reprogramming; Theriogenology; Reproduction, Fertility and Development; Molecular Reproduction and Development; Plos ONE; and Reproduction in Domestic Animals. These journals have published 37.9% of all published papers. The most frequent JCR categories in these journals are "reproductive biology", "veterinary sciences", "developmental biology", and "agricultural dairy animal sciences", and therefore, these journals are the most suitable for consulting or publishing on this area of research.

Discussion
In the present study, 124 papers on the reproduction of sheep by applying SCNT were counted in the WOS database; this quantity of papers was similar to that reported in a review carried out for the 25th anniversary of cloning using SCNT. This review published a survey of all published papers (1997-2020) on SCNT classified by species, among which mice, cattle, pigs, goats, and sheep were the species for which more than 100 published papers had been registered [43].
China, Italy, England, and Scotland are the countries that have generated more than 50% of the scientific research on the reproduction of sheep using SCNT. China is the world's leading producer of sheep, while Australia, Iran, and the United Kingdom (which includes England and Scotland) are among the ten countries with the highest number of sheep worldwide, according to FAO data for 2020 [44]. This could partly explain the interest of these countries in research on the reproduction of sheep by cloning.
The universities that have shown interest in the reproduction of sheep by cloning, are the University of Nottingham (England), the Roslin Institute of the University of Edinburgh (Scotland), the University of Teramo (Italy), and the China Agricultural University (China), which are the affiliations from the most cited authors in this area of research.
The principal journals that have published topics related to sheep reproduction by applying SCNT are Cellular Reprogramming; Theriogenology; Reproduction, Fertility and Development; Molecular Reproduction and Development; Plos ONE; and Reproduction in Domestic Animals. These journals have published 37.9% of all published papers. The most frequent JCR categories in these journals are "reproductive biology", "veterinary sciences", "developmental biology", and "agricultural dairy animal sciences", and therefore, these journals are the most suitable for consulting or publishing on this area of research.
The most cited article reports the birth of Dolly [21], obtained from a somatic cell from the mammary gland of an adult sheep, an unusual fact that had not been achieved in an upper mammal and which was a watershed for science, and therefore, for this reason, this article is still widely cited today. The other two most cited articles address issues about the generation of transgenic lambs for the production of human proteins for therapeutic purposes, which means that cell lines expressing a specific gene in the SCNT can be used, and this ensures that a lamb is obtained with the desired modification [25,26].
The words "oocyte" and "somatic cell" were frequent, and both cell types were used for SCNT. Oocytes have been treated with caffeine, which increases the activity of the promoter factor of maturation and of mitogen-activated protein kinases that are important for the nuclear reprogramming process during SCNT embryo development [33,45,46]. Moreover, the words "embryo", "gene expression", and "in vitro" were used in connection with each other. Examples include studies that have evaluated the effect of different chemical agents on the state of DNA methylation [47,48] and inhibitors of histone acetyltransferases [40,49] in the development of in vitro cloned embryos, especially at the blastocyst stage.
A detailed analysis of Figure 4 shows that cluster one, in red, contains 14 words related to the competence of in vitro cloned embryos. Khan et al. [50] compared the efficiency of conventional SCNT and handmade cloning for generating cloned sheep embryos. Better rates of efficiency of enucleation and fusion were obtained with handmade cloning, as well as a higher percentage of segmented and blastocyst stage embryos.
Cluster two, in bright green, contains 13 words referring to the development efficiency of cloned embryos at the blastocyst stage using different types of cells as donors of genetic information, most commonly cumulus cells and fibroblasts. The cells were treated with egg extracts [51], zebularine [47], chaetocin [52], and histone demethylase enzymes [53] to promote nuclear reprogramming of somatic cells and to improve the epigenetic status of ovine cloned embryos.
Cluster three, in blue, contains nine words focusing on the fetal development of cloned sheep and their regulation of gene expression. Ni et al. [54] evaluated the pregnancies of transgenic lambs produced by SCNT. They observed that fetal weight, total placenta weight, and mean placentomes weight were greater in pregnancies with live-born lambs, but did not survive compared to pregnancies with live-born lambs that survived. Further deregulation was found in miR-21 and miR-16 in the placenta of non-surviving lambs, causing aberrant expression in their targets.
Cluster four, in light green, contains nine words referring to the epigenetic status of cloned sheep embryos. Morphological evaluation is routinely used as the main parameter of embryo quality. However, due to the limited information provided by its unique evaluation, other parameters have focused on evaluating epigenetics [27,55] and the genetic status of sheep embryos generated by SCNT [9,52].
Finally, cluster five, in purple, includes five words related to the generation of transgenic sheep mediated cloning. Zhang et al. [56] investigated the effect of suppressing the myostatin (MSTN) gene expression in sheep skeletal muscle satellite cell using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9)) technology, to generate lamb clones with better muscle conformation; it has been reported that MSTN gene is responsible for regulating the growth of muscle cells.
It has been 26 years since the first sheep was cloned by applying SCNT, and since then, it has been applied to different domestic and wild species, in some cases with viable newborns. However, SCNT is an inefficient biotechnology, for example, Sheep were one of the first species to be domesticated for newborns lambs have been reported to be from 5.7 to 15% per transferred blastocyst and from 7.1% to 19.5% per segmented embryo [57]. Although sheep are easy to handle and have a relatively short gestation period compared with that of the other species of zootechnical interest [58], their small value and limited potential, do not make them attractive for agricultural use as compared with other livestock species [59]. If we also take into account that the infrastructure used for SCNT is basically the same for all species, nowadays, SCNT applied to sheep reproduction is not profitable. Therefore, the present bibliometric study shows the areas of research in which cloning research in sheep should be directed, which will help those regions where sheep are an important economic and food source.

Conclusions
Bibliometric studies on SCNT in sheep have not been conducted prior to now. This study collected bibliographic data from 124 papers relating to the application of SCNT in sheep. This amount of information about sheep is smaller than that for other species of zootechnical interest, such as cattle and pigs, although sheep were the first large mammal to be successfully cloned. Since 2001, the number of SCNT-related papers that have been published concerning ovine reproduction has increased and has fluctuated in ensuing years. The authors that have generated more knowledge in this area are (in alphabetical order) Campbell, Hajian, Hosseini, Loi, Nasr-Esfahani, Ptak, and Wilmut. Five research groups were identified, three of which mutually collaborated. The countries with the largest number of published papers were China, Italy, and England. The largest collaboration network among countries comprises China, Iran, Australia, Canada, and United States. The institutions with the highest productivity of SCNT in sheep are the University of Nottingham and the Roslin Institute at the University of Edinburgh. These two institutions are among the top 150 universities in the world. The principal journals where topics about SCNT in sheep are published have an IF ranging from 1.9 to 4.1, whose quartile position is most often in third and fourth place in the JCR thematic categories. These journals are the most suitable for publishing scientific advances in this area. The articles that have been cited more often have addressed topics related to the generation of transgenic animals, recovery of wild species, and xenotransplants. Five main themes were identified in sheep reproduction by SCNT. These themes focused on the competence of in vitro clone embryos, cells used as karyoplasts and their efficiency on embryo development, epigenetic status of clone embryos and their impact on post-implantation development, and generation of transgenic sheep with biomedical and genetic improvement purposes. Concerning the application of SCNT in sheep, these topics are the most relevant, and future studies should focus on solutions to the current challenges in this field of study. Institutional Review Board Statement: The present study was a bibliometric analysis that consisted of the use of databases of scientific papers. The study did not involve humans or animals.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available from first author José Roberto Vazquez-Avendaño (robertmizer@gmail.com) upon request.

Conflicts of Interest:
The authors declare no conflict of interest.