In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS-citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5+/-0.7% at an average sort rate of 1945.5+/-187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6+/-3.9%, 48.1+/-3.3%, 59.4+/-3.8% after DNA labelling, and 64.8+/-3.2%, 58.0+/-5.0%, 70.8+/-4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4 degrees C, sperm integrity, progressive and total motility were 46.4+/-5.2%, 32.2+/-4.2% and 58.2+/-3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen.

Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white (Ceratotherium simum), black (Diceros bicornis), Indian (Rhinoceros unicornis)] and both elephant species, the Asian and the African elephant (Elephas maximus, Loxodonta Africana), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants.

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2–3% in the freezing media, cooling rates of −30 to −50 °C/min, and thawing rates of 1200–1800 °C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1 × 109 spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.

Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number of species. However, the fertility of sex-sorted sperm is reduced and the full effects of sperm-sexing remain to be elucidated. The purpose of the present study was to investigate the potential effects of sex-sorted sperm on mRNA expression patterns of developmentally important genes employing in vitro produced bovine embryos. Bovine embryos were produced in vitro with unsorted and sex-sorted sperm and mRNA expression patterns were determined for glucose-3 transporter (Glut-3), glucose-6-phosphate dehydrogenase (G6PD), X-inactive specific transcript (X-ist) and Heat shock protein 70.1 (Hsp) using semi-quantitative endpoint reverse transcriptase-PCR in male and female, day-7 and 8 embryos. The relative abundance (RA) of Glut-3 was higher for day-7 male than female embryos, and day-7 embryos derived from unsorted compared with sex-sorted sperm. The RA of G6PD was higher for embryos derived from unsorted than sex-sorted sperm, and for day-8 female compared with male embryos. The RA of Xist was higher for female than male embryos, and for day-7 female embryos derived from unsorted than sex-sorted sperm. Hsp RA was higher for female compared with male embryos, was similar for day-7 and 8 embryos, and unsorted and sex-sorted sperm derived embryos. These results demonstrate differential expression of developmentally important genes between male and female embryos, and embryos derived from unsorted and sex-sorted sperm.

The aim of the present study was to ascertain whether multiparous sows could successfully be inseminated with sexed semen non-surgically. Spermatozoa were stained with Hoechst 33342 and separated flowcytometrically in X- and Y-chromosome bearing sperm populations employing the Beltsville Sperm Sexing Technology (BSST). After weaning, estrus was induced in sows with PMSG and hCG. Animals were inseminated once per estrus non-surgically with a specially designed catheter into the tip of the uterine horn, employing 50 × 106 of either sexed or non-sexed spermatozoa diluted in 2 ml Androhep™. Pregnant sows were allowed to go to term. Mean pregnancy rate from inseminations with unsexed spermatozoa was 54.5% whereas inseminations with sexed spermatozoa resulted in 33.3% pregnant sows. All but one piglet born after insemination with sexed semen were of the predicted sex. The sex of those piglets born after inseminations with non-sexed spermatozoa was 61.1% for male and 38.9% for female sex. It is concluded that non-surgically inseminations with flowcytometrically sexed spermatozoa can be conducted successfully.

Veterinary RecordVolume 152, Issue 13 p. 400-401 Short Communication Birth of female piglets following intrauterine insemination of a sow using flow cytometrically sexed boar semen D. Rath DrMedVethabil, D. Rath DrMedVethabil Institute of Ainimal Science and Animal Behaviour, Mariensee (FAL), 31535 Newstadt, GermanySearch for more papers by this authorS. Ruiz DVM, PhD, S. Ruiz DVM, PhD Department of Physiology, Institute of Biology, University of Murcia, Murcia, SpainSearch for more papers by this authorB. Sieg RBTA, B. Sieg RBTA Institute of Ainimal Science and Animal Behaviour, Mariensee (FAL), 31535 Newstadt, GermanySearch for more papers by this author D. Rath DrMedVethabil, D. Rath DrMedVethabil Institute of Ainimal Science and Animal Behaviour, Mariensee (FAL), 31535 Newstadt, GermanySearch for more papers by this authorS. Ruiz DVM, PhD, S. Ruiz DVM, PhD Department of Physiology, Institute of Biology, University of Murcia, Murcia, SpainSearch for more papers by this authorB. Sieg RBTA, B. Sieg RBTA Institute of Ainimal Science and Animal Behaviour, Mariensee (FAL), 31535 Newstadt, GermanySearch for more papers by this author First published: 29 March 2003 https://doi.org/10.1136/vr.152.13.400Citations: 14Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume152, Issue13March 2003Pages 400-401 RelatedInformation

Infertility is a common disease, which causes many couples to seek treatment with assisted reproduction techniques. Many factors contribute to successful assisted reproduction technique outcomes. One important factor is laboratory environment and air quality. Our facility had the unique opportunity to compare consecutively used, but separate assisted reproduction technique laboratories, as a result of a required move. Environmental conditions were improved by strategic engineering designs. All other aspects of the IVF laboratory, including equipment, physicians, embryologists, nursing staff and protocols, were kept constant between facilities. Air quality testing showed improved air quality at the new IVF site. Embryo implantation (32.4% versus 24.3%; P < 0.01) and live birth (39.3% versus 31.8%, P < 0.05) were significantly increased in the new facility compared with the old facility. More patients met clinical criteria and underwent mandatory single embryo transfer on day 5 leading to both a reduction in multiple gestation pregnancies and increased numbers of vitrified embryos per patient with supernumerary embryos available. Improvements in IVF laboratory conditions and air quality had profound positive effects on laboratory measures and patient outcomes. This study further strengthens the importance of the laboratory environment and air quality in the success of an IVF programme.