Special Issue "Biotechnology and Reproduction in Companion Animals"

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

Deadline for manuscript submissions: 30 November 2021.

Special Issue Editor

Dr. Sabine Schäfer-Somi
E-Mail Website
Guest Editor
Centre for Artificial Insemination and Embryo Transfer, University of Veterinary Medicine Vienna, 1210 Vienna, Austria
Interests: biotechnology; gamete preservation; spermatology; neonatology; immunology of pregnancy

Special Issue Information

Dear Colleagues,

Reproduction in small animals is an often overlooked field of veterinary science. The activities of scientific groups all over the world are increasing, covering all aspects of reproduction. It is a challenge to provide regular updates and comprehensive reviews. In particular, in the field of biotechnology, knowledge regarding gamete preservation, IVF, embryo transfer, cloning and so on is continuously increasing and tools such as open access publishing facilitate the exchange and sharing of valuable results. Biotechnology may carry negative associations, such as the belief that it is “unnatural”; however, in particular, endangered wild animal species benefit from the efforts of conservation medicine. This Special Issue presents a collection of state of the art reviews and original research articles, comprising andrological and gynecological themes as well as different aspects of ART and gamete preservation.

Dr. Sabine Schäfer-Somi
Guest Editor

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 papers will be 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 monthly 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 1800 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.

Keywords

  • reproduction
  • ART
  • embryo transfer
  • IVF
  • cloning
  • gamete preservation
  • spermatozoa
  • oocytes

Published Papers (1 paper)

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Research

Communication
Application of the FISH Technique to Visualize Sex Chromosomes in Domestic Cat Spermatozoa
Animals 2021, 11(7), 2106; https://doi.org/10.3390/ani11072106 - 15 Jul 2021
Viewed by 883
Abstract
Fluorescence in situ hybridization is a molecular cytogenetics technique that enables the visualization of chromosomes in cells via fluorescently labeled molecular probes specific to selected chromosomes. Despite difficulties in carrying out the FISH technique on sperm, related to the need for proper nuclear [...] Read more.
Fluorescence in situ hybridization is a molecular cytogenetics technique that enables the visualization of chromosomes in cells via fluorescently labeled molecular probes specific to selected chromosomes. Despite difficulties in carrying out the FISH technique on sperm, related to the need for proper nuclear chromatin decondensation, this technique has already been used to visualize chromosomes in human, mouse, cattle, swine, horse, and dog spermatozoa. Until now, FISH has not been performed on domestic cat sperm; therefore, the aim of this study was to visualize sex chromosomes in domestic cat sperm. The results showed the presence of X and Y chromosomes in feline spermatozoa. The procedure used for sperm decondensation and fluorescence in situ hybridization was adequate to visualize chromosomes in domestic cat spermatozoa and, in the future, it may be used to determine the degree of chromosomal abnormalities in these gametes. Full article
(This article belongs to the Special Issue Biotechnology and Reproduction in Companion Animals)
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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Title: Serum Antimullerian Hormone: a potential semen quality biomarker in stud dogs?
Authors: Eline Wydooghe, Guillaume Domain, Fien Uyttersprot, Osvaldo Bogado Pascottini, Joke Lannoo, Ann Van Soom
Affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
Abstract: Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance, has been known to be involved in the differentia-tion of the male reproductive tract during prenatal development. After birth, AMH is produced by Sertoli cells in males and granulosa cells in females. In dogs, the diagnostic value of measuring serum AMH concentrations to check the gonadal status has been well established (confirmation of cryptorchidism or presence of ovarian remnants). Recently, AMH has received increasing attention in research on male infertility in humans, albeit with contradictory results and conclusions. To date, no research has focused yet on the clinical applicability of AMH measurements in relation to semen quality in dogs. This study aims to investi-gate if there is an association between the concentration of serum AMH and the semen quality. In order to do so, 30 stud dogs were split in two groups based on their semen quality: i) good semen quality (progressive motility (PM) ≥70% and normal morphology (NM) ≥80% and viability≥ 90%) and intermediate to poor semen quality (PM <50% and/or NM <60% and/or viability <50%). In addition, the serum AMH concentration of 5 non-obstructive azoospermic dogs was measured.

Title: Freezability of dog semen after collection in field conditions and cooled transport.
Authors: Martina Colombo1, Maria Giorgia Morselli1, Giulia Franchi1§, and Gaia Cecilia Luvoni1
Affiliation: 1Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare “Carlo Cantoni”, Università degli Studi di Milano, Italy;
Abstract: The aim of this study was to evaluate whether freezing dog semen after 24 or 48 hr of cooled transport to an equipped laboratory is a valuable option when semen collection is performed in the field as in local breeding kennels, or during expositions. Ejacu-lated spermatozoa from 20 healthy stud dogs (mixed breeds, 2-13 years old) were collected by digital manipulation. One aliquot was evaluated as fresh control (FRESH) and one immediately cryopreserved (CRYO T0) by Uppsala method, whereas other two aliquots were diluted (1:1) with chilling or freezing extender (TRIS buffer, antibiotics, 20% egg yolk and 3% Glycerol only in freezing extender) and kept in a Minitube® styrofoam transport box for 24 hr (CRYO T24) or 48 hr (CRYO T48), until cryopreser-vation. Freezing of freshly collected semen is the optimal way to ensure a good sperm survival. However, results on motility, mor-phology, membrane and acrosome integrity of frozen spermatozoa, together with the evaluation of the ability of spermatozoa to adhere to oocytes zona pellucida will allow to verify whether a prolonged cooled transport before freezing could be proposed to dog breeders which cannot reach the lab with their animals for semen collection.

Title: Single layer centrifugation with Canicoll prior to cryopreserva-tion increases thawed semen quality in dogs
Authors: Guillaume Domain1, Ann Van Soom1, Osvaldo Bogado1, Anders Johannisson2, Jane Morrell2 and Eline Wydooghe1
Affiliation: 1 Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium 2 Department of Clinical Sciences, Division of Reproduction, Swedish University of Agricultural Sciences, 756 51 Uppsala, Sweden
Abstract: The aim of this study was (i) to assess the effect of sperm selection by single layer centrifugation (SLC) with Canicoll on semen freezability in dogs, and (ii) to evaluate whether this treatment had a similar effect regardless of the initial semen quality. Semen from 18 dogs was collected by digital manipulation and assigned to 2 groups : good quality semen (n=9, progressive motility ≥ 70% and normal morphology ≥ 80%) or medium quality semen (n=9, 40% ≤ progressive motility < 70% and/or 60% ≤ normal morphology < 80%). After collection, ejaculates were split in half and subjected to control centrifugation or SLC before being cryopreserved following a standard protocol. Assessment of sperm motility and velocity (computer-assisted sperm analysis), morphology (eosin-nigrosin staining), plasma membrane integrity, acrosome integrity and mitochondrial membrane potential (triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/JC-1), lipid peroxida-tion (induced TBARS assay) and DNA integrity (SCSA acridin orange) were performed on fresh and frozen-thawed samples. Sperm motility, velocity, morphology and plasma membrane integrity were significantly (P < 0.05) improved after SLC in both groups. However, SLC treatment significantly (P < 0.05) reduced the yield of high quality sperm collected prior to freezing in comparison with normal centrifugation (39.7 ± 6.5% vs. 48 ± 10.9% and 26.9 ± 10.3% vs. 30.3 ± 8.5%, for good and medium quality respectively). After thawing, sperm velocity, morphology and plasma membrane integrity of the SLC-treated samples were still significantly (P < 0.05) increased in both groups. In addition, mitochondrial membrane potential was significantly (P < 0.05) in-creased in SLC-treated samples of good quality. From these partial results, we can conclude that SLC with Canicoll is an effective treatment to select a subpopulation of sperm of higher quality regardless of the initial semen quality in dogs. Also, this in-creased semen quality can still be noticed after thawing. To prove that the fertility potential of this sperm has not been affected, inseminations of frozen-thawed SLC-selected sperm will be performed in a limited number of females.

Title: Reliability of a new portable device (iSperm®) for on-field as-sessment of sperm motility and concentration in dogs
Authors: Guillaume Domain, Ann Van Soom, Osvaldo Bogado, Joke Lannoo and Eline Wydooghe
Affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
Abstract: The iSperm has recently been released as an easy and affordable semen analysis tool which transforms an iPad Mini into a handheld microscope. The advantage of this device is that it can be used for on-field fresh sperm analysis, unlike a comput-er-assisted sperm analysis (CASA) device, which requires semen to be collected and analysed at the computer location. Initially developed for farm animals, a software for canine semen has now become available. The aim of this study is to assess the relia-bility of this new software in comparison with a validated CASA system. Three experiments will be conducted with the goal to (i) compare sperm motility and velocity between iSperm and CASA devices on both fresh and frozen-thawed samples, (ii) eval-uate whether the 4 different fields examined by the iSperm are comparable and (iii) assess the ability of the iSperm to estimate sperm concentration. Semen from 150 dogs was collected and results will be analysed for differences between the means and agreement will be investigated by computing Lin’s concordance correlation coefficient (CCC).

Title: A simplified procedure of hypoosmotic swelling test (HOST) for feline spermatozoa
Authors: Sylwia PROCHOWSKA1, Wojciech NIŻAŃSKI1, Alain FONTBONNE2
Affiliation: 1)Department of Reproduction and Clinic of Farm Animals, Wroclaw University of Environmental and Life Sciences pl. Grunwaldzki 49, 50-366 Wrocław; (2) L'École nationale vétérinaire d'Alfort, 7 Avenue du Général de Gaulle, 94700 Maisons-Alfort, France
Abstract: The hypo-osmotic swelling test (HOST) is used for the assessment of functional integrity of the sperm plasma membranes in many species. It has been shown for canine spermatozoa, that HOST can be easily and reliably performed in distilled water for 5 min. The aim was to optimize HOST procedure for the evaluation of feline semen in clinical conditions. Urethral semen was collected from 13 male domestic cats. HOST was performed by adding 5μL of semen to 50 μL of distilled water (0 mOsmol) or 50 mOsmol fructose and incubating samples at 37°C or room temperature for 5 and 30 min. After incubation, 200 spermatozoa were evaluated and those with coiled tails were considered HOST positive (functional membranes). Friedman ANOVA was performed for detecting the combined effect between the osmolarity, temperature and time. No statistically significant differences were found between the variables evaluated. Conclusions. HOST can be reliably performed in 0 mOsmol for 5 min at room temperature. Keywords: cat, spermatozoa, hypoosmotic swelling test, clinical practice This study was supported by statutory research and development activity funds assigned to Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences

Title: Prognostic markers for freezability of canine semen- in use and fiction
Authors: Schäfer-Somi S.
Affiliation: Besamungs- und Embryotransferstation, Vetmeduni Wien, Veterinärplatz 1
Abstract: To foresee freezability of canine semen is a challenge. Canine spermatozoa usually show good adaptability to changing osmolarity and temperature, however, in some individuals, the quality of the thawed semen is significantly worse despite good raw semen quality. To some extent the latter seems to depend on the constitution of subgroups of spermatozoa with variable kinetic characteristics and variable adaptability to osmolarity. Membrane fluidity is influenced by changes in the seminal plasma composition, composition and osmolarity of extenders, cooling/freezing/thawing protocols and may be genetics. Trials to influence one or the other of these parameters may cause membrane damages itsself, especially the fragile acrosome membrane with important enzymes and proteins like sperm-oocyte binding proteins, progesterone receptors and other factors, essential for capacitation and fertilization. Even with proven and standardized protocols, the quality of thawed canine semen is highly variable. There are different approaches to predict the freezability of raw canine semen. Easiest is to evaluate specific kinetic parameters by using a Computer Assisted Sperm Analyzer (CASA). Expression of the ABC transporter ABCA1 in spermatozoa membranes was related to freezability but the technique is elaborate. Functional tests like volumetry are also elaborate but highly predictive. Still under investigation for prediction of freezability of canine semen is the precursor of the A-kinase anchor protein 4 (ProAKAP4). In seminal plasma, low cholesterol content, high viscosity and low citric acid concentration proved to be detrimental for canine sperm freezing. Investigation of oocyte-sperm binding proteins and sperm surface proteins are promising; proteomic studies are upcoming but not yet available in dogs. Recent studies investigate the influence of genetics on sperm quality. Sperm quality, membrane integrity and fertility are supposed to be associated with certain single nucleotide polymorphisms (SNPs) allowing genotyping. Molecular markers allow selection for good freezers.

Title: Feline and canine testicular tissue preservation
Authors: Lúcia Daniel Machado da Silva
Affiliation: Faculty of Veterinary Medicine, State University of Ceará, Fortaleza-CE, Brazil
Abstract: Abstract The increased interest in breeding dogs and cats and their use as models for other canids and felids demand research to improve reproductive techniques. Among them, testicular cryopreservation stands out. Testicular cryopreservation enables the maintenance of reproductive capacity and allows the establishment of germplasm banks for several species of commercial value or at risk of extinction. Furthermore, it also enables the transport of genetic material between different regions. It is noteworthy that this biotechnology represents the only possibility to preserve the fertility of prepubertal animals that have died, being of great importance in the propagation of the genetic material of animals. The spermatogonia present in the testes can be cultivated in vitro and the sperm obtained used in artificial reproduction programs. Although some advances have been achieved with the use of testicular tissue to obtain viable and functional germ cells, the establishment of protocols that can be used in clinical routine have not yet been achieved. The testicular cryopreservation process can be carried out through techniques such as slow freezing, fast freezing and vitrification, however, the protocols used for the canine and feline species are still in the experimental phase. Given the importance of the topic, the aim of this review is to draw a profile on the subject, approaching the main works on testicular cryopreservation in dogs and cats.

Title: Canine cloning and mesenchymal stem cell: current and future directions in reproductive biotechnology
Authors: Eun Pyo Kim1,2, Hyun Ju Oh1,3, Byeong Chun Lee1, *, Kihae Ra1, *
Affiliation: 1 Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, 08826, Korea 2 Naeun Animal Hospital, Uiejeongbu, 11718, Korea 3 Division of Animal & Dairy Science, College of Agriculture and Life Science, Chungnam National University, Daejeon 34134, Korea * Correspondence: B.C.L. and K.R.
Abstract: Cloning is the process of generating individuals using somatic cells with identical DNA. Since the birth of the world's first cloned dog Snuppy, somatic cell nuclear transfer technology (SCNT) has been continuously developed as a part of advanced assisted reproduction technology and used in canine cloning. SCNT has been used to clone endangered canine species, working dogs, and companion dogs, all of which show normality in their health condition, growth characteristics, temperament, and behavior. To improve its efficiency, mesenchymal stem cells that have been recently introduced in assisted reproduction can be applied in the process of SCNT. Together with mesenchymal stem cell as reproductive intervention, advances in canine cloning using SCNT are highly expected. This review introduces the development of canine cloning technology and the current application of mesenchymal stem cells and their derivatives in reproductive biotechnology, further suggesting the possibility of their synergetic effects in canine cloning research.

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