title: Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa.
abstract: The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.
authors.affiliation: Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Lindsey
authors.forename: J L
authors.initials: JL
authors.lastname: Schenk
authors.forename: J K
authors.initials: JK
authors.lastname: Graham
authors.forename: J E
authors.initials: JE
authors.lastname: Bruemmer
authors.forename: E L
authors.initials: EL
authors.lastname: Squires
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000662:veterinary*; D005260:Female; D005298:Fertility / Q000502:physiology; D005434:Flow Cytometry / Q000662:veterinary*; D005615:Freezing; D006736:Horses / Q000502:physiology*; D015907:Hysteroscopy / Q000662:veterinary; D007315:Insemination, Artificial / Q000379:methods / Q000662:veterinary*; D008297:Male; D011247:Pregnancy; D018873:Pregnancy Rate; D011897:Random Allocation; D012662:Semen Preservation / Q000662:veterinary*; D013094:Spermatozoa / Q000502:physiology*