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A special issue of Animals (ISSN 2076-2615). This special issue belongs to the section "Animal Reproduction".

Deadline for manuscript submissions: closed (29 February 2024) | Viewed by 17137

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Dr. João Pedro Barbas

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Instituto Nacional de Investigação Agrária e Veterinária, Quinta da Fonte Boa, 2005-048 Vale de Santarém, Portugal
Interests: small ruminants; reproduction physiology and biotechnologies; andrology; reproductive efficiency and methods; reproduction parameters; sperm cryopreservation (new extenders, techniques, new methods, etc.); cervical artificial insemination
Special Issues, Collections and Topics in MDPI journals

Dr. Luis Águila Paredes

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Guest Editor

Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile
Interests: in vitro embryo production; early embryo development; embryonic pluripotency; developmental epigenetics; micromanipulation of gametes and embryos; ICSI; SCNT
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Recently, there have been increasing advances in “Reproductive Biotechnologies” seeking to optimize reproductive efficiency, productivity, gamete selection and preservation and health performance under current challenging environmental conditions. During recent decades, reproductive technologies have opened new avenues for studying and manipulating reproductive biology in different mammalian species with important economical, societal, and medical implications. In human medicine, one of the main applications is the treatment of infertility, but in animal reproduction, there are other applications. Among these are wildlife and domestic cell conservation, the improvement and preservation of animal genetics, and the enhancement of reproductive efficiency. Reproductive biotechnologies include artificial insemination, embryo transfer, estrus synchronization and superovulation, multiple ovulation embryo transfer, laparoscopic ovum pick-up, laparoscopic insemination, the in vitro production of embryos, intracytoplasmic sperm injection, the refrigeration and cryopreservation of sperm, the cryopreservation of oocytes and embryos, the sexing of sperm and embryos, embryo splitting, cloning and gene transfer, the production of artificial gametes and embryos and marker-assisted selection. That is why we are pleased to invite you to this Special Issue to publish high-quality research papers, review articles and communications addressing new advances in the field of reproductive biotechnologies in mammals.

The research topics may include (but are not limited to) the following: reproductive health; infertility; early embryo development and pregnancy; progress in molecular markers associated with fertility, gamete and embryo quality, and reproductive disorders, as well as designing breeding strategies; assisted reproductive technologies such as cryopreservation, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT), the micromanipulation of gametes and embryos, the genetic and genomic analysis of gametes and embryos, embryo biopsy, embryo transfer, fertility preservation, artificial insemination, oocyte and embryo culture, genomic selection and manipulation, transgenesis, transcriptomics and epigenomics, embryo outgrowths and embryonic stem cells; and the improvement of methods and techniques for gamete evaluation and reproductive efficiency methods.

Original, high-quality contributions that are not yet published or under review by other journals are sought. We hope that this issue will provide novel insights into the recent advancements in the field of reproductive biotechnologies, as well in basic and applied reproduction science.

We look forward to receiving your contributions.

Dr. João Pedro Barbas
Dr. Luis Águila Paredes
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Animals is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2400 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Published Papers (9 papers)

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18 pages, 4714 KiB

Open AccessArticle

Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium

by Moisés N. Segunda, Carlos Díaz, Cristian G. Torres, Víctor H. Parraguez, Mónica De los Reyes and Oscar A. Peralta

Animals 2024, 14(5), 803; https://doi.org/10.3390/ani14050803 - 05 Mar 2024

Viewed by 622

Abstract

In vitro gamete derivation has been proposed as an interesting strategy for treatment of infertility, improvement of genetic traits, and conservation of endangered animals. Spermatogonial stem cells (SSCs) are primary candidates for in vitro gamete derivation; however, recently, mesenchymal stem cells (MSCs) have also been proposed as candidates for germ cell (GCs) differentiation mainly due to their transdifferentiating capacity. The objective of the present study was to compare the potential for GC differentiation of bovine peripheral blood-derived MSCs (PB-MSCs) and SSCs under the effect of conditioned medium (CM) derived from Sertoli cells (SCs/CM). Samples were collected every 7 days for 21 days and analyzed for pluripotent, GC, and MSC marker expression. The absence of OCT4 and the increased (p < 0.05) expression of NANOG seems to play a role in SSC differentiation, whereas the absence of NANOG and the increased expression (p < 0.05) of OCT4 may be required for PB-MSC differentiation into GCs. SSCs cultured with SCs/CM increased (p < 0.05) the expression of PIWIL2 and DAZL, while PB-MSCs cultured under the same condition only increased (p < 0.05) the expression of DAZL. Overall, the patterns of markers expression suggest that PB-MSCs and SSCs activate different signaling pathways after exposure to SCs/CM and during differentiation into GCs. Full article

(This article belongs to the Special Issue Recent Advances in Reproductive Biotechnologies)

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13 pages, 3933 KiB

Open AccessArticle

Is the Hippo Pathway Effector Yes-Associated Protein a Potential Key Player of Dairy Cattle Cystic Ovarian Disease Pathogenesis?

by Esdras Corrêa Dos Santos, Alexandre Boyer, Guillaume St-Jean, Natalia Jakuc, Nicolas Gévry, Christopher A. Price and Gustavo Zamberlam

Animals 2023, 13(18), 2851; https://doi.org/10.3390/ani13182851 - 08 Sep 2023

Viewed by 910

Abstract

Cystic ovarian disease (COD) in dairy cattle is characterized by preovulatory follicles that become cysts, fail to ovulate and persist in the ovary; consequently, interfering with normal ovarian cyclicity. The intraovarian key players that orchestrate the alterations occurring in the preovulatory follicle and that culminate with cyst formation and persistence, however, remain uncertain. Interestingly, the Hippo pathway effector yes-associated protein (YAP) has been described in humans and mice as a key player of anovulatory cystic disorders. To start elucidating if YAP deregulation in ovarian follicle cells can be also involved in the pathogenesis of COD, we have generated a series of novel results using spontaneously occurring cystic follicles in cattle. We found that mRNA and protein levels of YAP are significantly higher in granulosa (GCs) and theca cells (TCs) isolated from cystic follicles (follicular structures of at least 20 mm in diameter) in comparison to respective cell types isolated from non-cystic large follicles (≥12 mm). In addition, immunohistochemistry and Western blot analyses used to determine YAP phosphorylation pattern suggest that YAP transcriptional activity is augmented is cystic GCs. These results were confirmed by a significant increase in the mRNA levels encoding for the classic YAP-TEAD transcriptional target genes CTGF, BIRC5 and ANKRD1 in GCs from follicle cysts in comparison to non-cystic large follicles. Taken together, these results provide considerable insight of a completely novel signaling pathway that seems to play an important role in ovarian cystic disease pathogenesis in dairy cattle. Full article

(This article belongs to the Special Issue Recent Advances in Reproductive Biotechnologies)

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15 pages, 2486 KiB

Open AccessArticle

Effect of Atomized Black Maca (Lepidium meyenii) Supplementation in the Cryopreservation of Alpaca (Vicugna pacos) Epididymal Spermatozoa

by Gloria Levano, Juana Quispe, Diego Vargas, Marlon García, Alberto López, Luis Aguila and Martha Valdivia

Animals 2023, 13(13), 2054; https://doi.org/10.3390/ani13132054 - 21 Jun 2023

Cited by 1 | Viewed by 2304

Abstract

Artificial insemination is an important assisted reproductive technology that has been applied in several mammalian species. However, successful cryopreservation of semen of South American camelids has been limited, hindering the commercial application of artificial insemination in alpaca species. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve the freezability of mammalian sperm. Bioactive metabolites from natural extracts of black maca have shown valuable antioxidant properties. Thus, the objective of this study was to evaluate the effect of the addition of atomized black maca in the freezing medium of epididymal spermatozoa of alpacas. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. Markers associated with functionality, such as motility, viability, and plasma membrane integrity, as well as markers associated with oxidative damage, such as DNA integrity, total ROS production, and mitochondrial function, were analyzed. The results show that the supplementation with black maca (20 mg/mL) improved the sperm motility, viability, plasma membrane integrity, and mitochondrial function evaluated according to an index of formazan deposits. Similarly, the ROS production decreased with maca at 20 mg/mL, although the DNA integrity did not show any differences among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers (proAKAP4) associated with fertility. Full article

(This article belongs to the Special Issue Recent Advances in Reproductive Biotechnologies)

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14 pages, 2102 KiB

Open AccessArticle

Administration of Beta-Nerve Growth Factor during the Preovulatory Stage Improves Endocrine and Luteal Function in Dairy Heifers

by Gonzalo Gajardo, Luis Paiva, Cesar Ulloa-Leal, Ximena Valderrama, Gerardo López, Albert Carrasco, Alejandra Isabel Hidalgo, Mauricio E. Silva, Patricio I. Palma and Marcelo H. Ratto

Animals 2023, 13(6), 1004; https://doi.org/10.3390/ani13061004 - 09 Mar 2023

Cited by 1 | Viewed by 1353

Abstract

The neurotrophin beta-nerve growth factor (NGF), which is present in the semen of different mammals, elicits potent ovulatory and luteotrophic actions in llamas following systemic administration. Here, we determine if purified NGF given intramuscularly (IM) during the preovulatory stage affects the corpus luteum (CL), hormone production, endometrial gene expression, and pregnancy rate of dairy heifers. Holstein-Friesian heifers were estrus-synchronized using estradiol benzoate (EB) plus an intravaginal progesterone (P4) device (DIB). After eight days, the device was removed and cloprostenol was given IM; the next day (day 9), heifers received EB IM plus one of the following: (i) 1 mg of NGF (NGF D9 group), (ii) 1 mg of NGF 32 h after EB (NGF D10 group), or (iii) phosphate buffer saline (control group). To measure pregnancy rates, heifers were treated similarly, then artificially inseminated with sexed semen 48–52 h after DIB removal, then an ultrasound was conducted 30 days after insemination. The females given NGF along with EB (NGF D9) showed significantly higher luteinizing hormone (LH) concentrations, larger CL vascular areas, and higher plasma P4 concentrations than the NGF D10 and control animals. Downregulation of the P4 receptor (PGR), and upregulation of both lipoprotein lipase (LPL) and Solute Carrier Family 6 member 14 (SLC6A14) endometrial genes, were detected in NGF D9 heifers. Furthermore, these heifers had a 10% higher pregnancy rate than the control group. We conclude that the higher P4 output, in response to the early NGF administration, led to the enhanced gene expression of transcripts related to uterine receptivity that may result in enhanced pregnancy rates. Full article

(This article belongs to the Special Issue Recent Advances in Reproductive Biotechnologies)

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14 pages, 765 KiB

Open AccessArticle

Ram Semen Cryopreservation for Portuguese Native Breeds: Season and Breed Effects on Semen Quality Variation

by João Pedro Barbas, Jorge Pimenta, Maria Conceição Baptista, Carla Cruz Marques, Rosa Maria Lino Neto Pereira, Nuno Carolino and João Simões

Animals 2023, 13(4), 579; https://doi.org/10.3390/ani13040579 - 07 Feb 2023

Cited by 3 | Viewed by 2030

Abstract

The semen quality is one of the determinant factors of ram semen cryopreservation. The present retrospective study aimed to characterize the seasonal ram pattern during the year for ten Portuguese local sheep breeds, hypothesizing that the breed and season had low effects on the main spermatozoa traits. A total of 1471 ejaculates were used and evaluated (fresh semen) from 85 rams between 2004 and 2020 and re-evaluated after thawing (thawed semen). The effect of breed, season, and sperm cryopreservation on nine semen traits were evaluated. The volume per ejaculate, spermatozoa (SPZ) concentration, and total number of SPZ per ejaculate, were affected by breed (p < 0.001) but not by season (p > 0.05). As expected, the semen processing was the most significant (p < 0.001) factor of variation on seminal parameters. Moreover, breed and interactions between breed × semen processing, modulated the response of alive SPZ, abnormal morphology, head, and intermediate piece defects. In fresh semen, season only affected the intermediate piece defects due to the highest percentage observed between February and April period in some breeds. Overall, and despite the mentioned particularities, there were similarities among the ten local breeds. We also concluded that the seasonal effect on ejaculate and SPZ traits is not significant in our region. These local ram breeds have low seasonality and can be employed in natural mating as well as semen donors for cryopreservation and assisted reproductive biotechnologies during the whole year at our latitude. Full article

(This article belongs to the Special Issue Recent Advances in Reproductive Biotechnologies)

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20 pages, 13269 KiB

Open AccessArticle

Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC)

by Moisés N. Segunda, Carlos Díaz, Cristian G. Torres, Víctor H. Parraguez, Mónica De los Reyes and Oscar A. Peralta

Animals 2023, 13(2), 318; https://doi.org/10.3390/ani13020318 - 16 Jan 2023

Cited by 2 | Viewed by 1971

Abstract

Although spermatogonial stem cells (SSC) constitute primary candidates for in vitro germ cell (GC) derivation, they are scarce and difficult to maintain in an undifferentiated state. Alternatively, mesenchymal stem cells (MSC) are also candidates for GC derivation due to their simplicity for culture and multipotential for transdifferentiation. The aim of the present study was to compare the GC differentiation potentials of bull peripheral blood-derived MSC (PB-MSC) and SSC using an in vitro 3D co-culture system with Sertoli cells (SC). Samples of PB-MSC or SSC co-cultures with SC were collected on days 0, 7, 14 and 21 and analyzed for pluripotency, GC and mesenchymal marker expression. Co-culture of PB-MSC+SC resulted in down-regulation of NANOG and up-regulation of OCT4 at day 7. In comparison, co-culture of SSC+SC resulted in consistent expression of NANOG, OCT4 and SOX2 at day 14. During co-culture, SSC+SC increased the expression of DAZL, PIWIL2, FRAGILIS and STELLA and activated the expression of STRA8, whereas co-culture of PB-MSC+SC only increased the expression of DAZL and PIWIL2. Thus, co-culture of bull PB-MSC+SC and SSC+SC in 3D SACS results in differential expression of pluripotency and GC markers, where bull SSC display a more robust GC differentiation profile compared to PB-MSC. Full article

(This article belongs to the Special Issue Recent Advances in Reproductive Biotechnologies)

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13 pages, 666 KiB

Open AccessArticle

Simple and Efficient Chemically Defined In Vitro Maturation and Embryo Culture System for Bovine Embryos

by María Elena Arias, Tamara Vargas, Victor Gallardo, Luis Aguila and Ricardo Felmer

Animals 2022, 12(21), 3057; https://doi.org/10.3390/ani12213057 - 07 Nov 2022

Cited by 5 | Viewed by 2469

Abstract

Supplementation of the culture media for in vitro production (IVP) of bovine embryos with fetal bovine serum (FBS) is associated with inconsistent outcomes. The present study sought to replace FBS and BSA by insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). In Experiment 1, absence of FBS from maturation medium (MM) did not affect the rate of in vitro maturation, as assessed by the extrusion of the first polar body. However, when gonadotropins and FBS were removed from the MM, the maturation rate was significantly reduced even in the presence of growth factors. Therefore, gonadotropin-supplemented MM medium was established as the base medium for the defined maturation condition. In Experiment 2, the addition of growth factors to gonadotropin-supplemented MM medium supported similar maturation (~90%) compared to the undefined condition (FBS-carrying). In Experiment 3, the addition of growth factors to embryo culture medium showed similar in vitro competence compared to the undefined (FBS) control. In Experiment 4, completely defined conditions (absence of FBS and BSA during in vitro maturation and embryo culture) were tested. A higher cleavage was observed with FGF2 (86%) compared to EGF (77%) and the FBS control (77%), but similar blastocyst rates were observed for FGF2 (24%), EGF (19%) and the FBS control (25%). Embryo quality was similar among groups. Finally, post-thawing survival was higher for FGF2 (94%) compared to the FBS control (77%). Thus, we report a simple defined IVP system for bovine species that generates developmental outcomes and embryos of similar quality than those produced under conditions containing FBS. Full article

(This article belongs to the Special Issue Recent Advances in Reproductive Biotechnologies)

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17 pages, 2719 KiB

Open AccessArticle

Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations

by Jahaira Cortez, Barbara Leiva, Cristian G. Torres, Víctor H. Parraguez, Mónica De los Reyes, Albert Carrasco and Oscar A. Peralta

Animals 2022, 12(17), 2283; https://doi.org/10.3390/ani12172283 - 03 Sep 2022

Cited by 7 | Viewed by 1983

Abstract

Organoids are 3D-culture systems composed of tissue-specific primary cells that self-organize and self-renew, creating structures similar to those of their tissue of origin. Testicular organoids (TOs) may recreate conditions of the testicular niche in domestic and wild cattle; however, no previous TO studies have been reported in the bovine species. Thus, in the present study, we sought to generate and characterize bovine TOs derived from primary testicular cell populations including Leydig, Sertoli and peritubular myoid cells. Testicular cells were isolated from bovine testes and cultured in ultra-low attachment (ULA) plates and Matrigel. TOs were cultured in media supplemented from day 3 with 100 ng/mL of BMP4 and 10 ng/mL of FGF2 and from day 7 with 15 ng/mL of GDNF. Testicular cells were able to generate TOs after 3 days of culture. The cells positive for STAR (Leydig) and COL1A (peritubular myoid) decreased (p < 0.05), whereas cells positive for WT1 (Sertoli) increased (p < 0.05) in TOs during a 28-day culture period. The levels of testosterone in media increased (p < 0.05) at day 28 of culture. Thus, testicular cells isolated from bovine testes were able to generate TOs under in vitro conditions. These bovine TOs have steroidogenic activity characterized by the production of testosterone. Full article

(This article belongs to the Special Issue Recent Advances in Reproductive Biotechnologies)

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12 pages, 5462 KiB

Open AccessCase Report

Surgical Description of Laparoscopic Ovum Pick-Up in Buffalo Calves

by Alysson J. de O. Sousa, Heytor J. Gurgel, Paula S. A. Coelho, Carla R. G. Silva, Luiz H. V. Araújo, Hamilton S. do Nascimento, Izamara do S. R. Rodrigues, Luciano C. Pantoja, Thiago da S. Cardoso, Maykon D. Silva, Ana Carolina C. Torres, Pedro Paulo M. Teixeira and Moysés dos S. Miranda

Animals 2023, 13(1), 102; https://doi.org/10.3390/ani13010102 - 27 Dec 2022

Viewed by 1991

Abstract

The technique of laparoscopic oocyte aspiration has been increasingly used in animals; however, there are few records of its use in buffaloes. To describe this technique, six suckling Murrah buffaloes aged between 3 and 5 months were used. Three laparoscopic ovum pick-ups were performed in each animal, with intervals of 15 days between surgeries, completing a total of 18 procedures. The technique used three surgical ports with optics and a high-definition video camera. The introduction of the first portal and insufflation of the abdomen was performed through the open technique, with aspiration using a 20 G needle transabdominally and a vacuum pump calibrated at 50 mmHg. The mean complete surgical time from anesthesia to the removal of the animal from the litter was 49 ± 9.8 min. There were 27.8% cases of insufflation on the wrong side of the omentum. The oocyte recovery rate of 60.3% remained within the normal range. However, the rate of viable oocytes recovered was low, with only 40.8% of those recovered undergoing in vitro embryo production (IVEP). These data demonstrate that this simple, minimally invasive technique is an excellent reproductive tool for the genetic improvement of buffalo species. Full article

(This article belongs to the Special Issue Recent Advances in Reproductive Biotechnologies)

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