1. 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.
4. 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 (1997–2006) 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.
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)/CRISPR-associated 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.
5. 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.